host factors regulating retroviral replication by
TRANSCRIPT
Host factors regulating retroviral replication
by interactions with viral RNA and DNA
Gary Zhe Wang
Submitted in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
under the Executive Committee
of the Graduate School of Arts and Sciences
COLUMBIA UNIVERSITY
2016
© 2016
Gary Zhe Wang
All Rights Reserved
ABSTRACT
Host factors regulating retroviral replication
by interactions with viral RNA and DNA
Gary Zhe Wang
Retroviruses are capable of infecting diverse vertebrates, and successful infection requires
intimate interaction between virus and the host cell. During an infection, retroviral particles must
bind specifically to cell surface receptors on the target cell, cross the plasma membrane, reverse-
transcribe their RNA genome into double stranded DNA, find their way to the nucleus, enter the
nucleus and integrate its DNA into host chromosomes. Following integration, expression of viral
mRNA ensues, followed by viral mRNA export into the cytoplasm, translation of viral mRNA
into proteins, and assembly of new virions that will egress from the host cell. We now appreciate
that at many steps of this complex process, the virus must hijack the cellular machinery to
replicate. At the same time, the host cell mobilizes a variety of cellular defense mechanisms to
suppress viral infection. This thesis investigates various aspects of virus-host interactions. I will
first describe the involvement of cellular transcriptional repressor protein ErbB3 binding protein
1 (EBP1) in facilitating transcriptional shutdown of Moloney murine leukemia virus (MLV) gene
expression in mouse embryonic cells. Next, I describe a novel means of regulating the activity of
Yin Yang 1 (YY1), a cellular transcription factor regulating retroviral gene expression, through
post-translational modifications. I show that YY1 is a target of tyrosine phosphorylation by Src
family kinases. Phosphorylation of YY1 impairs its ability to bind DNA and RNA, thereby
downregulating its activity as a transcription factor on retroviral and cellular promoters. Apart
from studying retroviral gene expression, I have also investigated intrinsic cellular defenses
against retroviral infection. This is exemplified by our finding that mouse cells are intrinsically
resistant to infection by betaretroviruses such as Mason-Pfizer monkey virus (M-PMV). The
block against M-PMV occurs after reverse transcription and prior to viral nuclear entry. Finally, I
will present ongoing work examining the fate of viral DNAs following infection, focusing on the
kinetics of its association with cellular core histones and viral structural proteins. Together, this
work provides critical insights into numerous aspects of the virus-host interactions.
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TABLE OF CONTENTS
LIST OF FIGURES ...................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................ iii
CHAPTER 1 - INTRODUCTION ............................................................................................... 1
Overview of the retroviral life cycle ......................................................................................................... 1
Yin Yang 1 (YY1) .................................................................................................................................. 14
Cross-species restriction of retroviruses ................................................................................................. 17
Concluding remarks – setting the stage for projects addressing virus-host interactions at multiple steps
of the early viral life cycle ...................................................................................................................... 19
CHAPTER 2 – EBP1, A NOVEL HOST FACTOR INVOLVED IN PRIMER BINDING
SITE-DEPENDENT RESTRICTION IN EMBRYONIC CELLS ........................................ 20
CHAPTER 3 – REGULATION OF YIN YANG 1 BY TYROSINE PHOSPHORYLATION
....................................................................................................................................................... 25
CHAPTER 4 – POSTENTRY RESTRICTION OF MASON-PFIZER MONKEY VIRUS
IN MOUSE CELLS .................................................................................................................... 36
CHAPTER 5 – HISTONES ARE RAPIDLY LOADED ONTO UNINTEGRATED
RETROVIRAL DNAS SOON AFTER NUCLEAR ENTRY ................................................. 43
CHAPTER 6 - DISCUSSION .................................................................................................... 81
EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney murine
leukemia virus in embryonic cells .......................................................................................................... 81
Regulation of Yin Yang 1 by Tyrosine Phosphorylation ........................................................................ 84
Postentry restriction of Mason-Pfizer monkey virus in mouse cells ....................................................... 88
Histones are rapidly loaded onto unintegrated retroviral DNAs soon after nuclear entry ...................... 90
REFERENCES ............................................................................................................................ 94
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LIST OF FIGURES Figure 1-1: The genome architecture of retroviruses .................................................................................... 2
Figure 1-2: The retroviral life cycle .............................................................................................................. 3
Figure 1-3: Modes of viral entry into the host cell ....................................................................................... 5
Figure 1-4: Retroviral reverse transcription reaction .................................................................................... 6
Figure 1-5: Modes of viral nuclear entry ...................................................................................................... 8
Figure 1-6: Viral integration reaction ........................................................................................................... 9
Figure 1-7: Mechanism of MLV silencing in mouse embryonic cells ........................................................ 13
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank my advisor, Dr. Stephen Goff, for his mentorship,
guidance and support. Steve is a phenomenal scientist, a creative thinker, a role model and
someone who truly cares about his trainees. When I first joined the lab, I was a naïve,
overzealous graduate student who wants to work on a new project every day. Steve gave me the
freedom to pursue my ideas. When experiments didn’t work as I envisioned, I would knock on
his door and say “Hey boss, you busy, or can we talk about some science?”, and the answer
would always be “sure, come on in”. This open-door policy saved my butt time and time again. I
have learned so much from him over the past four years. As I write this thesis, I no longer want
to work on a new project every day. I now know the importance of positive and negative controls.
I am now a lot more critical of my own data as well as the data I see. I now know when to
keeping chasing the unknown and when to ditch the project. I now know to “blow off” 2-fold
effects as noise. I now understand the need to address the same question from multiple angles. I
now know when to “emphasize!!!” key findings and when to be cautious and modest about what
I say. I could go on and on. Most importantly, I now know that I love science and I want to be a
researcher for the rest of my life.
I want to thank my thesis advisory committee, Dr. Richard Baer, Dr. Carol Prives, Dr.
Vincent Racaniello for their time and expertise. I also would like to thank Dr. Uttiya Basu and
Dr. Paul Bieniasz from Rockefeller University for their time and input in my thesis defense.
Thank you all for holding my hand during what I think is the toughest degree of my life.
I had a blast being a member of the Goff lab. I could not have done what I have done
without their intellectual input, sharing reagents, and a fun working environment. Thanks to Dr.
Sharon Schlesinger for introducing me to the world of epigenetics and mouse embryonic cells.
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Thanks to my bench-mate Yossi Sabo for all the stimulated discussions, inappropriate jokes and
advice about raising kids. Thanks to Dr. Yiping Zhu for giving me all the tips on how to make
biochemistry look easy. Thanks to Dr. Daniel Griffin for all the clinical insights and stories about
being a sailor. Thanks to Dr. Angela Erazo for helpful discussions and making “Taco Tuesdays”
a Goff lab tradition. Thanks to Dr. Michael Metzger for advice on PCR/cloning/old school
molecular biology, and recommendations on “must-do’s” when visiting Seattle. Thanks to Dr.
Oya Cingӧz for introducing me to the world of innate immunity. Thanks to all my fellow
students in the lab (Andreia Lee, Cheng Wang, Sedef Onal, Marina Ermakova, Marlene Arroyo)
for student-to-student advice. Thanks to Kenia de los Santos, Martha de los Santos and Helen
Wang for putting up with my excessive lab orders and reagent requests. Thanks to Martine
Lecorps for helping me with all the administrative hassles during graduate school.
Thanks to Dr. Ron Liem, Dr. Michael Shelanski, Dr. Steven Reiner, Dr. Patrice Spitalnik,
Mrs. Zaia Sivo and everyone in the MD/PhD program for giving me a chance to train at
Columbia and live in New York City during the prime time of my life.
Thanks to my father Jim Wang, my mother Helen Liu for their love and understanding
throughout my life. Thanks to my wife Ying Wang and my daughter Emily Wang for endless
support and joy. This work is dedicated to my family.
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CHAPTER 1 - INTRODUCTION
Overview of the retroviral life cycle
Retroviruses are single-stranded positive-sense RNA viruses capable of infecting many
vertebrates. Infection typically results in life-long chronic viremia. The key to their persistence is
the integration of proviral DNA into the genome of the host’s somatic and germ-line cells.
Infection can cause cell death and lead to depletion of the target cell population; or more often
can eventually lead to leukemia by insertional mutagenesis.
A retroviral virion contains two copies of the single-stranded RNA genome, which
encodes the viral proteins required for productive infection. All known retroviruses contain three
major genes called “gag’, “pol”, and “env’, which encode structural proteins (gag), enzymes
such as reverse transcriptase and protease (pol), as well as surface and transmembrane proteins
(env) (Fig. 1-1). The viral genes in the context of the integrated viral DNA are flanked on either
end by repeated nucleic acid sequences termed long terminal repeats (LTR). LTRs play
indispensable roles in the retroviral life cycle, including viral integration into the host genome
and regulation of viral gene expression.
The life cycle of retroviruses can be divided into two phases (Fig. 1-2). The early phase
begins with binding of virions to cell surface receptors and ends with integration of viral DNA
into the host chromosomes. The late phase begins with the expression of viral genes and ends
with the maturation and release of new virions from the cell surface.
In the first step of the retroviral life cycle, viral envelope glycoproteins interact with
cognate cellular surface receptors. For example, ecotropic Moloney murine leukemia virus
(MLV) utilizes a ubiquitously expressed mouse amino acid transporter (mCAT) to gain entry
into many cell types (Kim et al, 1991; Wang et al, 1991), whereas human immunodeficiency
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Figure 1-1: The genome architecture of retroviruses
The representative genome of Moloney murine leukemia virus (MLV) is shown. Viral genes,
gag, pol, and env are flanked on both ends by LTR sequences.
Diargram adopted from http://viralzone.expasy.org/all_by_species/67.html.
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Figure 1-2: The retroviral life cycle
Retroviral life cycle can be divided into two phases. Early phase begins with virion binding to
cell surface receptors and ends with integration. Late phase begins with transcription of viral
genes in the nucleus and ends with viral maturation and budding.
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virus-1 (HIV-1) utilizes the CD4 protein of T-lymphocytes as the receptor (Maddon et al, 1986).
The presence or absence of a particular cell surface receptor defines the virus’s cell-type tropism.
Receptor attachment is followed by entry of the virus into the host cell. Different viral envelopes
mediate virus entry into the target cell via distinct mechanisms (Fig. 1-3). For example, the
glycoprotein from vesicular stomatitis virus (VSV-G) promotes viral entry via endocytosis, while
amphotropic (ampho) MLV envelope and HIV-1 envelope facilitate direct fusion with the
plasma membrane (McClure et al, 1990). In the case of ecotropic (eco) MLV envelope, there
exists evidence consistent with both endocytosis (i.e. pH dependence) and direct cell fusion (i.e.
pH independence) (Mothes et al, 2000; Nussbaum et al, 1993).
Following cell entry, the viral core, containing the RNA genome and its associated
proteins, is released into the cytoplasm of the target cell and undergoes partial uncoating to give
rise to a ribonucleoprotein complex termed the reverse transcription complex (RTC). With the
help of the viral-encoded reverse transcriptase enzyme, the single-stranded viral RNA genome is
converted into double-stranded viral DNA genome in a stepwise manner (Fig. 1-4). First, cellular
tRNA bound to the primer binding site (PBS) of the viral genomic RNA functions to prime DNA
synthesis by reverse transcriptase. This is followed by strand extension and strand transfer
reactions, leading to the generation of full length viral DNA genomes. What triggers the
initiation of reverse transcription is not completely understood. One possibility is that exposure
of RTC to the presence of deoxyribonucleotides in the cytoplasm of the host cell may be
sufficient to drive reverse transcription (Goff, 2001).
Following reverse transcription, viral DNA genomes undergo trafficking toward the
nucleus of the infected cell. Given that the cellular cytoplasm is densely populated with proteins
and organelles, random diffusion of viral particles toward the nucleus may be an inefficient mean
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Figure 1-3: Modes of viral entry into the host cell
Different viral envelopes enter the cell by distinct mechanisms. The glycoprotein of vesicular
stomatitis virus (VSV-G) enters the cell by first binding to cell surface receptors. Endocytosis
and subsequent exposure of the virus to the low endosomal pH triggers the fusion of the viral
membrane with the endosomal membrane, this is termed pH-dependent entry. In contrast, the
envelope protein of amphotropic MLV, or HIV-1 can bind to cell surface receptors and directly
fuse with the cell membrane, this is termed pH-independent entry.
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Figure 1-4: Retroviral reverse transcription reaction
1. Using host tRNA as a primer, minus-strand DNA synthesis proceeds until the 5’ end of the
genomic RNA is reached. This short DNA intermediate is termed minus-strand strong-stop DNA
(sss-DNA).
2. RNase-H degrades the RNA strand of the RNA-sssDNA duplex. sssDNA undergoes strand
transfer and anneals to the 3’ end of the viral genomic RNA.
3. Minus-strand DNA synthesis resumes, while RNase H digestion of the RNA template strand
occurs. Note that all of the RNA template is degraded by RNase H with the exception of a
short stretch of RNA called polypurine tract (PPT).
4. The remaining PPT portion of the viral RNA genome serves to prime plus-strand DNA
synthesis.
5. RNase H removes the primer tRNA, and sssDNA anneals to the complementary PBS
segment in minus-strand DNA, this process is called second strand transfer.
6. Plus and minus-strand synthesis is complete.
Diagram is adopted from (Coffin et al, 1997).
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of reaching the target. Consequently, retroviruses have evolved means to hijack cellular
cytoskeletal machinery to facilitate purposeful movements toward the cell nucleus. For
example, HIV-1 infection induces the formation of stable microtubules in the infected cell,
which functions as specialized tracks for HIV-1 particle transport toward the nucleus (Sabo et
al, 2013).
The nuclear envelope of infected cells poses a formidable barrier to retroviral nuclear
translocation (Fig. 1-5). Indeed, retroviruses such as MLV are unable to penetrate intact nuclei of
infected cells, and must therefore depend on breakdown of the nuclear envelope during mitosis
to gain nuclear entry (Roe et al, 1993). In contrast, lentiviruses such as HIV-1 are able to actively
transverse the nuclear envelope of non-dividing cells such as resting T-lymphocytes and
macrophages (Weinberg et al, 1991). Here, certain HIV-1 proteins interact with
nucleocytoplasmic shuttling receptors called importins to gain nuclear entry in a manner similar
to cellular proteins.
Once inside the nucleus, the viral encoded integrase enzyme catalyzes the insertion of
linear viral DNAs into the host chromosomes (Fig. 1-6). The integration reaction proceeds in two
steps. First, integrase catalyzes the removal of two nucleotides from the 3’ terminus of the viral
LTR, termed 3’-end processing (Brown et al, 1989; Roth et al, 1989). The resultant viral DNA
substrate is then joined to the target DNA on host chromosomes through a strand transfer
reaction (Craigie et al, 1990; Engelman et al, 1991). Structurally, the catalytic pocket of integrase
from many different retroviruses contain a centrally located D-D(35)-E motif. For MLV,
mutations in the D-D(35)-E motif (e.g. D184A) results in the loss of all catalytic activity, making
it a useful tool to study integration independent viral processes (Lai et al, 2001).
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Figure 1-5: Modes of viral nuclear entry
Different viruses enter the nucleus of the infected cell by different mechanisms. To gain access
to host chromosomes, MLV requires the dissolution of nuclear envelope during cell division. In
contrast, HIV-1 can be actively imported into the nucleus by interaction with nuclear pore
complexes, rendering them capable of infecting non-dividing cells.
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Figure 1-6: Viral integration reaction
The integration reaction proceeds in two distinct steps. First, integrase catalyzes the removal of
two nucleotides from the 3’ terminus of the viral LTR, termed 3’-end processing. This exposes a
3’ hydroxyl group (OH), which acts as a nucleophile to attack target DNA during the joining
reaction. Joining reaction generates a DNA gap with 5’ 2-nucleotide overhangs, which can then
be removed, and the gap repaired to complete integration.
Diagram is adopted from (Coffin et al, 1997).
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Do retroviruses integrate randomly or at preferred sites in the host genome? Initial in
vitro studies demonstrated that proviral insertion occurs irrespective of DNA sequences but is
influenced by the chromatin structure (Pryciak et al, 1992). Gammaretroviruses such as MLV
prefers to integrate near transcription start sites, as well as in the vicinity of strong enhancers and
active promoters (De Ravin et al, 2014; LaFave et al, 2014; Wu et al, 2003). Lentiviruses
including HIV-1 prefer to integrate inside the bodies of active genes, especially genes that were
activated in cells following HIV-1 infection (Schroder et al, 2002). Thus, such strong bias in
integration site selection may promote robust retroviral gene expression.
Following integration, the viral promoter encoded in the LTR region functions to drive
the transcription of viral genes in a manner similar to cellular genes. The resultant viral RNA can
then be exported into the cytoplasm and be translated by host ribosomes to generate viral
proteins. Alternatively, full length viral RNA can also be packaged directly into progeny virions.
After assembly of viral RNA and proteins, new immature virions bud from the plasma
membrane and the Gag and Gag-Pol precursor proteins undergo proteolytic cleavage by the viral
protease, generating mature virions capable of infecting new cells.
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Transcriptional regulation of MLV in embryonic cells
MLV is unable to replicate in mouse embryonic stem (ES) or embryonic carcinoma (EC)
cells (Barklis et al, 1986; Teich et al, 1977). In embryonic cells, reverse transcription and
proviral integration proceed normally, but viral transcription is repressed, and hence no viral
gene products can be detected. Several mechanisms are likely involved in the transcriptional
silencing, including 1) the absence of enhancer proteins recognizing binding sites in the viral
LTR (Hilberg et al, 1987; Linney et al, 1984); 2) recruitment of trans-acting transcriptional
repressors (Akgun et al, 1991; Flanagan et al, 1989; Tsukiyama et al, 1989), and 3) de novo
DNA methylation (Niwa et al, 1983). One critical site for transcriptional silencing, termed the
repressor binding site (RBS), shows extensive overlap (17/18 bps) with the primer binding site
(PBSpro) of MLV (Barklis et al, 1986; Feuer et al, 1989; Loh et al, 1987), which normally
functions during the priming of reverse transcription by host proline tRNA. The RBS by itself is
sufficient to induce potent transcriptional repression of reporter constructs in EC cells,
irrespective of its orientation or position (Loh et al, 1990; Petersen et al, 1991). In addition,
electrophoretic mobility shift assays (EMSAs) using RBS as the probe demonstrate the presence
of stem-cell specific nuclear factors. A single base-pair mutation in the RBS, termed the B2
mutation, is sufficient to abolish nuclear factor binding, and thereby restore viral gene expression
(Loh et al, 1990; Petersen et al, 1991). These findings have allowed the characterization of the
stem-cell specific trans-acting transcriptional repressor complex as containing Trim28 (also
known as KAP-1 or Tif1-beta), a known transcriptional co-repressor (Wolf et al, 2008a; Wolf &
Goff, 2007; Wolf et al, 2008b), and Krüppel associated box (KRAB) zinc finger protein 809
(ZFP809) (Wolf & Goff, 2009), a DNA binding transcription factor. The current model for PBS-
mediated silencing consists of the binding of PBSpro by ZFP809, and the subsequent recruitment
of Trim28 to the provirus via the ZFP809 KRAB box domain. This association permits Trim28-
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dependent recruitment of additional chromatin modifiers such as the histone H3K9
methyltransferase SETDB1 (Schultz et al, 2002), heterochromatin-associated protein gamma
(HP1gamma) (Wolf et al, 2008a), the NURD histone deacetylases complex (Schultz et al, 2001)
and EBP1 (Wang et al, 2014). Together, the ensuing histone and DNA methylation of the
proviral DNA ensures transcriptional silencing and genomic stability (Fig. 1-7).
Beside the ZFP809/Trim28 complex, another zinc finger protein, Yin Yang 1 (YY1), also
binds to the LTR of MLV in embryonic cells, augmenting the recruitment of Trim28 and
downstream chromatin modifiers to the proviral DNA (Schlesinger et al, 2013). YY1 binding to
the viral LTR occurs rapidly following infection, leading to the initiation of MLV promoter
silencing. Indeed, retroviral gene expression is significantly upregulated upon MLV infection of
embryonic cells devoid of YY1, or by infecting wild-type embryonic cells with MLV LTR
mutant that no longer bind YY1. Therefore, both ZFP809 and YY1 are capable of binding
separate DNA sequences on the viral LTR and act to cooperatively recruit Trim28 and its
downstream epigenetic modifiers to turn off viral gene expression.
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Figure 1-7: Mechanism of MLV silencing in mouse embryonic cells
(A) In embryonic cells, two zinc finger transcription factors, YY1 and ZFP809 binds to unique
DNA sequences in the MLV LTR upon infection. Together, this leads to the recruitment of
Trim28, a host transcriptional repressor, as well as chromatin modifiers such as histone
methyltransferase SETDB1, NuRD histone deacetylases complex, HP1 and EBP1 to the proviral
promoter. These chromatin modifiers are responsible for the deposition of repressive histone
marks such as H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3) to
initiate MLV silencing. Ultimately, complete MLV silencing is achieved and maintained by
DNA methylation. (B) ZFP809 is not expressed in differentiated cells, hence no MLV silencing
is observed.
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Yin Yang 1 (YY1)
Yin Yang 1 (YY1) is a ubiquitously expressed DNA/RNA binding transcription factor
that takes its name from its dual activity on the adeno-associated virus (AAV) P5 promoter,
where the presence of adenoviral protein E1A converts YY1 from a transcriptional repressor to
an activator (Shi et al, 1991). In mice, targeted deletion of YY1 results in peri-implantation
lethality, indicative of an essential role in embryogenesis (Donohoe et al, 1999). YY1 binds to
DNA through the recognition of a specific consensus sequence, leading to the activation or
repression of many cellular genes including c-Myc, c-Fos, -actin, IFN-, CREB, Sp1 and others
(Gordon et al, 2006; Shi et al, 1997). Moreover, YY1 has been shown to physically interact with
numerous proteins required for cell growth, apoptosis and cancer progression, including p53,
Mdm2, Ezh2, and Rb (Gordon et al, 2006; Shi et al, 1997). In Xenopus oocytes, YY1 has been
demonstrated to associate with structurally divergent RNA species (Belak et al, 2008), raising
the possibility that YY1 can bind both DNA and RNA. Highlighting this dual-binding property,
YY1 was shown to tether Xist, a noncoding RNA (lncRNA), to DNA regions on the X
chromosome, leading to X chromosome inactivation in maternal cells (Jeon & Lee, 2011).
Therefore, it appears that the zinc finger domain of YY1 can simultaneously bind different
nucleic acid motifs in Xist RNA and DNA (Jeon & Lee, 2011).
Apart from its function on cellular promoters, YY1 plays a critical role in the
transcriptional regulation of retroviral promoters. For example, YY1 binds to and represses the
HIV-1 promoter, thereby maintaining the virus in a latent state (Bernhard et al, 2013; Margolis et
al, 1994). As discussed above, in the case of MLV, YY1 directly binds to the negative control
region (NCR) in the viral LTR (Flanagan et al, 1992), leading to transcriptional silencing of
MLV in mouse embryonic cells (Schlesinger et al, 2013). Consistent with its dual role in
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transcriptional activation, YY1 potentiates expression from the Human T-lymphotropic Virus 1
(HTLV-1) promoter, likely through association with the HTLV-1 RNA (Wang GZ and Goff SP,
unpublished observations).
The sequence-specific DNA binding activity of YY1 is mediated by four C2H2-type zinc
finger motifs (amino acid 298-414) located in the C-terminus (Hariharan et al, 1991). Besides
their role in DNA binding, portions of the zinc finger motifs (amino acid 333-397), together with
a region rich in alanine and glycine (amino acid 157-201) contributes to transcriptional
repression (Bushmeyer et al, 1995; Galvin & Shi, 1997). The N-terminal region contains several
unusual features also required for transcriptional activation. This includes two stretches of acidic
residues (amino acids 16-29 and amino acid 43-53), followed by a glycine rich region (amino
acid 54-69), 11 consecutive histidine residues (amino acid 70-80), and sequences rich in proline
and glutamine (amino acid 80-100) (Austen et al, 1997; Bushmeyer et al, 1995; Lee et al, 1995;
Lee et al, 1994).
Despite the wealth of knowledge garnered about YY1 interacting proteins and target
promoters regulated by YY1, much less is known about YY1 regulation. Based on previous
studies, YY1 appears to be a target of several posttranslational modifications, some of which
contribute to changes in YY1 activity. Sumoylation of YY1 by PIASy on lysine 288 negatively
affects the transcriptional activity of YY1 (Deng et al, 2007), while O-linked glycosylation of
YY1 disrupts its interaction with Rb, leading to enhanced DNA binding (Hiromura et al, 2003).
Moreover, YY1 also undergoes a complex acetylation/deacetylation cycle that changes its
affinity for DNA and capacity to interact with histone deacetylases (Yao et al, 2001). In prostate
cancer cells, nitric oxide (NO) has been shown to facilitate S-nitrosylation of YY1 on cysteine
residues, resulting in decreased DNA binding and upregulation of Fas expression (Hongo et al,
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2005). Serine/threonine phosphorylation of YY1 occurs during the cell cycle. Specifically,
threonine 39 is a target of Polo-like kinase 1 (Plk1), but the functional importance of this
modification remains to determined (Rizkallah et al, 2011b). Phosphorylation of serine 180 and
184 by Aurora B kinase has been shown to have effects on DNA binding and YY1’s ability to be
acetylated by histone acetyltransferase p300 (Kassardjian et al, 2012). Finally, phosphorylation
of threonine 348 and 378 in the DNA binding domain of YY1 by an unknown kinase leads to
decreased DNA binding (Rizkallah & Hurt, 2009).
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Cross-species restriction of retroviruses
Successful retroviral infection requires cellular factors to support each phase of the
retroviral life cycle. Therefore, retroviral tropism is partly dictated by whether the infected cell
possesses the necessary co-factors to assist viral replication. For example, HIV-1 infects human
T cells and macrophages efficiently but is unable to infect mouse cells. Multiple steps in the viral
life cycle are blocked in mouse cells. First, the mouse homologues of the human T cell receptors
and coreceptors CD4, CXCR4 and CCR5 (Atchison et al, 1996; Landau et al, 1988) cannot bind
the HIV env-encoded gp120, preventing HIV-1 from entering the cell. This entry block can be
bypassed in engineered mouse cell lines expressing the human T-cell receptors (Browning et al,
1997; Clayton et al, 1988), or alternatively, by pseudotyping HIV-1 virions with foreign viral
envelope proteins such as the glycoprotein G of the vesicular stomatitis virus (VSV-G). Upon
establishment of the provirus in mouse cells, the late phase of the HIV-1 life cycle is hindered by
additional cellular blocks. Transcription initiated in the Long Terminal Repeat (LTR) is defective
due to the incompatibility of the HIV-1 Tat transactivator with mouse cyclin T1 (Fujinaga et al,
1999; Garber et al, 1998; Kwak et al, 1999; Wei et al, 1998). In addition, viral assembly is
defective due to the absence of necessary factors required for late stages of the retroviral life
cycle, a phenotype that can be rescued by the fusion of uninfected human cells with infected
mouse cells (Bieniasz & Cullen, 2000; Mariani et al, 2001; Mariani et al, 2000). Study of blocks
to HIV-1 replication in mouse cells has led to the identification and characterization of critical
cellular factors necessary for infection.
Species-specific retroviral tropism is also governed by the presence of species-specific
restriction factors. For example, human and primates are known to be resistant to N-tropic MLV
infection (Besnier et al, 2003; Towers et al, 2000). The human protein mediating this restriction
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was identified as TRIM5 (Perron et al, 2004). In addition to restricting N-tropic MLV, rhesus
macaque TRIM5 has the remarkable ability to restrict HIV-1 infection (Stremlau et al, 2004;
Yap et al, 2004). This observation was later attributed to species-specific variations in the
TRIM5 sequence (Sawyer et al, 2005; Stremlau et al, 2005; Yap et al, 2005), supporting the
notion that species-specific restriction factors are a major determinant of retroviral tropism.
Mechanistically, TRIM5 has been proposed to interact with the viral capsid (CA) (Sebastian &
Luban, 2005), leading to premature dissociation of viral cores (Stremlau et al, 2006), and
terminating infection prior to nuclear entry (Stremlau et al, 2004). In mice, the Friend virus
susceptibility factor 1 (Fv1) gene functions similarly to TRIM5. Fv1 encodes a gag-like protein
with homologies to the ERV-L family of endogenous retroviruses (Best et al, 1996). There are
two major naturally occurring alleles of Fv1. NIH Swiss mice carry the Fv1n allele, which
restricts the replication of B-tropic MLVs but not N-tropic MLVs. In contrast, BALB/c mice
carry the Fv1b allele, which confers resistance to N-tropic MLV infection but not B-tropic MLVs
(Goff, 1996; Stoye, 1998). A few laboratory mice and some wild mice carry a third Fv1 allele,
Fv1nr
, which restricts the replication of B-tropic MLVs and a subset of N-tropic MLVs (Kozak,
1985; Pincus et al, 1971).
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Concluding remarks – setting the stage for projects addressing virus-host interactions at
multiple steps of the early viral life cycle
For the next few chapters, I will share my published work on different facets of the retroviral life
cycle. Chapter 2 and 3 focus on the regulation of gene expression from the MLV promoter in
mouse embryonic cells. In chapter 2, I will describe a novel ZFP809 interacting protein, EBP1,
and its involvement in potentiating MLV silencing in mouse embryonic cells. In chapter 3, I
discuss regulation of YY1 activity through tyrosine phosphorylation, and its implications in the
control of viral and cellular promoters. Chapters 4 and 5 will move away from retroviral gene
expression and focus on other early events of infection. In chapter 4, I describe the ability of
mouse cells to block infection by betaretroviruses such as M-PMV at the step of viral nuclear
entry. In chapter 5, I describe the most recent project examining the “state” of the viral DNAs
following infection, focusing on the association of viral DNAs with cellular histones during early
infection.
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CHAPTER 2 – EBP1, A NOVEL HOST FACTOR INVOLVED IN PRIMER BINDING
SITE-DEPENDENT RESTRICTION IN EMBRYONIC CELLS
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CHAPTER 3 – REGULATION OF YIN YANG 1 BY TYROSINE PHOSPHORYLATION
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CHAPTER 4 – POSTENTRY RESTRICTION OF MASON-PFIZER MONKEY VIRUS
IN MOUSE CELLS
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CHAPTER 5 – HISTONES ARE RAPIDLY LOADED ONTO UNINTEGRATED
RETROVIRAL DNAS SOON AFTER NUCLEAR ENTRY
Histones are rapidly loaded onto unintegrated retroviral DNAs
soon after nuclear entry
Gary Z. Wang1,2
, Ying Wang3, Stephen P. Goff#
4,5,6
1Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, New
York, NY 10032, USA
2Medical Scientist Training Program, Columbia University College of Physicians and Surgeons,
New York, NY 10032, USA
3 Division of Nephrology, Department of Medicine, Columbia University Medical Center, New
York, NY 10032, USA
4Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY
10032, USA
5Department of Microbiology and Immunology, Columbia University, New York, NY 10032,
USA
6Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA
Running title: Histone association with retroviral DNAs
Address correspondence to Stephen P. Goff
Abstract word count: 334
Text word count: 45208
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ABSTRACT
In the early stages of retroviral infection, a DNA copy of the viral genome is formed by
reverse transcription of the RNA genome. The course of DNA synthesis take place in the
cytoplasm in the context of a large structure closely resembling the virion core. The product of
this reaction is a fully double-stranded linear DNA contained within a pre-integration complex
(PIC), which retains many copies of the viral capsid (CA) and nucleocapsid (NC) proteins. The
linear viral DNA is delivered into the nucleus by routes that differ among virus strains. A portion
of the linear DNA then gives rise to circular double-stranded DNAs containing either one or two
copies of the Long Terminal Repeat (LTR) sequences, and a portion is inserted into the host
genome to form the integrated provirus. The proviral DNA eventually becomes covered with
nucleosomes, and depending on the cell type, the histone tails of the nucleosomes are modified
by the addition of histone marks that regulate the expression of the viral DNA. The timing of
nucleosome loading onto the viral DNA and, in particular, whether nucleosomes are loaded onto
unintegrated viral DNAs, has not been determined. In this study, we show that unintegrated viral
DNAs of Moloney murine leukemia virus rapidly associate with core histones. Loading of
nucleosomes on the viral DNA requires nuclear entry but does not require viral DNA integration.
Moreover, we observe that histone loading is correlated with unloading of viral structural
proteins from the viral DNA. The histones associated with unintegrated DNAs are marked by
covalent modifications, but the appearance of the modified histones is delayed relative to the
time of core histone loading. Expression from the unintegrated DNA can be enhanced by
modulation of the histone modifying machinery. Together, our data show that histones are
loaded on unintegrated DNAs very rapidly after nuclear entry and not only after formation of the
integrated provirus.
KEYWORDS: Retrovirus/Nucleosome/Histone/Epigenetics
45
INTRODUCTION
DNA of eukaryotic chromosomes is condensed into chromatin by the association with
nucleosomes, each consisting of an octomeric complex of the four core histones. Nucleosomes
are normally loaded onto chromosomal DNA during the course of DNA replication
(Ramachandran & Henikoff, 2015) . As a replication fork moves along the DNA, nucleosomes
are displaced from the unreplicated DNA ahead of the fork and then must reassociate onto both
daughter branches behind the growing fork. The current models suggest that the relocalization of
the nucleosomes is a highly dynamic process in which the “old” nucleosomes are distributed to
both daughter branches, and “new” nucleosomes are added to restore normal nucleosome density.
During this process, epigenetic marks on the old chromatin must be preserved or reestablished on
the new DNAs to ensure maintenance of the appropriate pattern of gene expression (Zhu &
Reinberg, 2011). New nucleosomes may also be loaded at sites of DNA damage, perhaps under
the direction of the PCNA factor present at the sites. The loading of nucleosomes is thought to be
promoted by an array of dedicated chaperones, including CAF-1, ATRX, and DAXX (Burgess &
Zhang, 2013). In contrast to these normal settings of chromatin formation, there are unusual
circumstances when naked DNA, or DNA previously free of nucleosomes, enters a cell and must
acquire a nucleosomal array de novo. In these situations there are no “old” or preexisting
nucleosomes to seed or facilitate the loading of new nucleosomes onto the DNA. One such
circumstance occurs in the course of retroviral infection.
Retroviruses are single-stranded RNA viruses with a complex life cycle. The core of the
mature virion particle consists of a protein shell made up of the viral capsid protein (CA)
enclosing two copies of the RNA genome tightly associated with the viral nucleocapsid protein
(NC). The viral NC protein is a small, highly basic protein that is tightly bound to the viral RNA
46
and condenses while the CA protein forms a hexameric lattice that encapsidates the viral RNA.
Following viral entry into the target cell, the single-stranded viral RNA genome is copied by
reverse transcriptase (RT) to generate a linear double-stranded DNA version of the genome,
contained within a large complex termed the preintegration complex (PIC). Following the
completion of reverse transcription, the double-stranded viral DNA and its associated proteins
are trafficked into the nucleus of the infected cell by one of a variety of mechanisms that vary
among different viral genera. For example, gammaretroviruses such as Moloney murine
leukemia virus (MLV) are unable to enter the intact nucleus of a nondividing cell and depend on
nuclear membrane breakdown during cell division to gain entry (Roe et al, 1993). In contrast,
lentiviruses such as HIV-1 are actively transported across the nuclear membrane even in non-
dividing cells such as macrophages and resting T lymphocytes (Weinberg et al, 1991). The
distinct requirements for nuclear entry also serve as a point of restriction for cross-species
transmission of retroviruses. For example, betaretroviruses such as Mason-Pfizer monkey virus
(M-PMV) are unable to enter the nucleus of mouse cells, defining a potent cross-species block at
this step of the viral life cycle (Wang & Goff, 2015).
Once inside the nucleus, viral encoded integrase (IN) enzyme catalyzes the insertion of
linear viral DNAs into the host chromosomes. The integration reaction proceeds in two steps.
First, IN catalyzes the removal of two nucleotides from the 3’ terminus of the viral long terminal
repeat (LTR), termed 3’-end processing (Brown et al, 1989; Roth et al, 1989). The resultant viral
DNA substrate is then joined to the target DNA on host chromosomes through a strand transfer
reaction (Craigie et al, 1990; Engelman et al, 1991). Structurally, the catalytic pocket of IN from
many different retroviruses contain a centrally located D-D(35)-E motif. For MLV, mutations in
47
the D-D(35)-E motif (e.g. D184A) results in the loss of all catalytic activities, making it a useful
tool to study integration independent viral processes (Lai et al, 2001).
Homologous recombination of linear viral DNAs at the LTR leads to the formation of 1-
LTR circles. 2-LTR circles, containing full length viral DNA and both sets of LTRs arranged
back to back, are products of non-homologous end joining (NHEJ) DNA repair pathways in the
nucleus. This property makes 2-LTR circles a useful marker of viral nuclear import, as the
unique nature of the LTR-LTR junction allow their levels to be readily quantified by PCR
(Butler et al, 2001).
In permissive cells, following integration, the 5’ LTR of the proviral DNA serves as the
promoter to generate full-length genomic viral mRNAs necessary for viral gene expression and
genome replication. However, certain cell types possess the ability to block this step of the
retroviral life cycle. For example, MLV is unable to replicate in mouse embryonic stem (ES) or
embryonic carcinoma (EC) cells (Akgun et al, 1991; Hilberg et al, 1987; Linney et al, 1984). In
embryonic cells, reverse transcription and proviral integration proceed normally, but viral
transcription is repressed, and hence no viral gene products can be detected. One major
mechanism in the transcriptional silencing of MLV in embryonic cells involves the recruitment
of trans-acting transcriptional repressors such as ZFP809, Trim28, YY1 and Ebp1 (Rowe et al,
2010; Schlesinger et al, 2013; Wang et al, 2014; Wolf & Goff, 2007; Wolf & Goff, 2009) to
DNA binding sites on the viral LTR. This results in the recruitment of chromatin modifiers such
as SETDB1 (Matsui et al, 2010), which deposits repressive histone marks such as H3K9
trimethylation (H3K9me3) en route to complete promoter silencing.
Several fundamental aspects of retroviral replication remain unexplored. Given that
reverse transcription generates de novo foreign DNAs in the host cell, this raises the question of
48
what is the histone state of these retroviral DNAs following infection. Are retroviral DNAs a
target of histone loading? If so, when, where and on what forms of viral DNA does this occur?
Following reverse transcription, do retroviral DNAs remain associated with their cognate
structural proteins such as NC and CA? When and where does NC and CA dissociate from the
viral DNA?
In this study, using MLV as a model, we describe the kinetics of histone association with
the viral DNA genomes. We show that unintegrated linear viral DNAs and 2-LTR circles
undergo rapid core histone loading in a nuclear entry-dependent and integration-independent
manner. Moreover, histone loading appears to be correlated with the removal of viral structural
proteins such as NC and CA from the viral DNA. In contrast to core histone loading, loading of
epigenetically modified histones occurs after a delay, thereby postponing host cell regulation of
the viral promoter. Finally, we show that unintegrated viral DNAs do give rise to a low level of
gene expression, and that this expression can be increased by changing the activity of chromatin
modifiers. Together, our data provide insights into fundamental aspects of retroviral biology.
49
RESULTS
Core histone loading onto retroviral DNAs occurs rapidly following infection
To investigate histone loading onto viral DNA genomes following infection, we utilized a
single-round MLV reporter genome in which viral genes were replaced with the GFP gene
(MLV-GFP). Virions capable of infecting a wide range of cell lines were prepared by collecting
culture medium after transfection of 293T cells with plasmids encoding the MLV-GFP reporter
genome, the MLV gag-pol protein, and the VSV-G envelope protein. This reporter virus is
capable of carrying out all the early events of a single round of infection, including reverse
transcription, nuclear entry, integration, and reporter gene expression. Kinetic studies
characterizing the formation of viral DNA replication intermediates following infection were
first performed. Mouse embryonic fibroblasts (MEFs) were transduced with MLV-GFP reporter
virus, total DNA was harvested at 12, 24 h or 6 days post infection, and levels of viral DNA
replication intermediates was monitored by quantitative real-time PCR using primers specific for
GFP (to detect total viral DNA) or 2-LTR circles (a marker of nuclear entry) (Fig. 1A). To
control for potential plasmid DNA carryover in the viral preparation, transduction of MEFs with
heat inactivated (HI) virus were carried out in parallel. Total viral DNA, which includes linear
DNA, circular DNA, and integrated proviral DNA, appeared at 12 h post infection, rose to a peak
at 24 h, and then decreased to a stable copy number of approximately 3200 copies by 6 days (Fig
1A). The 2-LTR circles appeared later, rose to a peak at 24 h post infection, and was completely
undetectable by 6 days, indicative of their short half-life in the nucleus (Fig. 1B). Of note, the
maximum amount of 2-LTR circles at early time points accounts for 10-20% of total viral DNAs.
As negative controls, MEFs transduced with heat-inactivated virus yielded no PCR signal,
indicating minimal contaminating plasmid DNA carryover from the virus preparation.
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We next monitored the association of nucleosomal histone H3 with retroviral DNAs by
Chromatin immunoprecipitation (ChIP). Infected MEF cells were formaldehyde cross-linked at
12, 24 h, and 6 days post infection, and processed to yield chromatin of appropriate size (200-
800 base pairs). Chromatin immunoprecipitation (ChIP) assays were performed with antibodies
specific for histone H3 or non-specific IgG control, followed by quantitative real-time PCR
analysis of immunoprecipitated DNA using primers targeting GFP (total viral DNA) or 2-LTR
circles. As a positive control, PCR primers targeting the promoter region of endogenous GAPDH
gene were used to represent fully chromatinized cellular DNA. Approximately 10% of the
GAPDH promoter sequences were specifically immunoprecipitated with the anti-histone H3
antibody, but not the non-specific IgG control antibody (Fig. 1B). Assuming that the GAPDH
promoter is fully chromatinized at all times, these results suggests that our ChIP recovery
efficiency is approximately 10%. In the case of viral DNAs, we detected rapid association of
histone H3 with total viral DNA to levels comparable to that of the cellular GAPDH gene by 24
h post infection (Fig. 1B). At 6 days, the observed ChIP signal with the GFP probe (total viral
DNA) remained high, indicative of fully chromatinized proviral DNA. In the case of 2-LTR
circles, histone H3 loading reached similar levels to that of the cellular GAPDH gene by 12 h
post infection (Fig. 1B). Of note, the signal of chromatin-bound DNA in the total DNA was too
high to be accounted for only by the circular DNAs, thusthe majority of the signal must be
derived from the linear form. Similar kinetics of histone loading onto viral DNAs were observed
in MLV-GFP infected F9 embryonic carcinoma cells (Fig. 1C and 1D). Together, these findings
suggest that both total and circular retroviral DNA forms undergo rapid chromatinization
following infection.
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Histone loading of retroviral DNAs occurs independently of viral integration
To investigate whether viral integration is needed for efficient histone loading, two
independent approaches were taken. First, MLV-GFP reporter viruses were packaged using wild-
type (WT) or catalytically inactive MLV integrase (IN) mutant (D184A) (Lai et al, 2001). The
infectivity of WT or D184A-IN virions was confirmed using flow cytometry analysis of infected
NIH-3T3 fibroblasts 48 h post infection (Fig. 2B). Compared to WT virus, a 70-fold reduction in
the infectivity of D184A-IN virions was observed (Fig. 2B). Moreover, cells infected with
D184A-IN virions contained slightly higher levels of total viral DNA (GFP) and 2-LTR circles at
12 and 24 h post infection, but no viral DNA was detected at 6 days post infection (Fig. 2C).
This suggests that the D184A mutation has no adverse effects on reverse transcription and viral
nuclear entry, but is defective in viral integration. Compared to WT virus, histone H3 ChIP of
cells infected with D184A-IN virus showed comparable accumulation of histone H3 on both
total viral DNA (GFP) and 2-LTR circles (Fig. 2D). This observation suggests that viral
integration is not required for histone loading and that most of the chromatinized viral DNA
early on likely represent unintegrated linear and circular DNA forms.
Alternatively, similar ChIP experiments were performed on MEF cells infected with WT-
virus in presence or absence of the integrase inhibitor raltegravir (Fig. 2E). As expected,
raltegravir treatment prevented the formation of GFP+ cells (Fig. 2F). Analogous to the case for
D184A-IN virus, raltegravir treatment caused a 2-fold increase in the levels of 2-LTR circles
without affecting total viral DNA (GFP) levels (Fig. 2G). Compared to mock treated cells, ChIP
analysis using raltegravir treated cells revealed normal accumulation of histone H3 on both total
viral DNA (GFP) and 2-LTR circles (Fig. 2H), further highlighting the non-requirement for
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integration. Taken together, our data suggests that early during infection, chromatinization of
total viral DNA occurs irrespective of viral integration.
Histone loading of retroviral DNAs requires viral nuclear entry
It is well established that 2-LTR circles, products of host cell non-homologous end
joining, are found exclusively in the nucleus, making it a useful marker for viral nuclear entry
(Bukrinsky et al, 1992; Bukrinsky et al, 1991; Shoemaker et al, 1980). Like many nuclear
proteins, histones are first synthesized in the cytosol and later trafficked to the nucleus (Burgess
& Zhang, 2013). Since viral reverse transcription also takes place in the cytoplasm and that
linear viral DNA is able to undergo rapid chromatinization following infection (Fig. 2D and 2H),
we examined the possibility that histone loading onto viral DNAs may occur in the cytoplasm
prior to viral nuclear entry. Previously, it has been shown that in mitotically active cells, nuclear
envelope breakdown facilitates entry of the MLV preintegration complex (PIC) into the nucleus,
while growth arrested cells that fail to undergo cell division block MLV infection at nuclear
entry (Roe et al, 1993). To block nuclear entry, NIH-3T3 cells were treated with or without
aphidicolin, a reversible inhibitor of eukaryotic DNA synthesis for 24 h (Fig. 3A). DNA content
analysis of aphidicolin treated cells using fluorescence activated cell sorting (FACS) revealed
cell cycle arrest in the G1/S phase (Fig. 3B). Growth arrested cells were then transduced with
MLV-GFP reporter viruses for 24 h, after which chromatin from infected cells was subjected to
ChIP using histone H3 antibodies. At 24 h post infection, aphidicolin treatment did not affect
levels of total viral DNA (GFP), hence suggesting that viral entry, uncoating and reverse
transcription proceeded normally (Fig. 3C). In contrast, a 30-fold reduction in the levels of 2-
LTR circles was observed, indicative of defective viral nuclear entry (Fig. 3C). Strikingly,
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despite normal levels of input total viral DNA, ChIP analysis revealed a 10-fold reduction in
histone H3 associated viral DNA in aphidicolin arrested cells, whereas histone H3 coverage of
the cellular GAPDH gene was unaffected by the drug (Fig. 3C).
To further address the nuclear entry requirement for retroviral histone loading, we took
advantage of our earlier finding that mouse cells exhibit a nuclear entry block to Mason-Pfizer
monkey virus (M-PMV) infection (Wang & Goff, 2015). Mouse Ba/F3 cells, a pro-B cell line,
were infected with VSV-G pseudotyped MLV-GFP or M-PMV-GFP (Fig. 3E), and the
efficiency of infection was monitored by flow cytometry analysis 48 h later (Fig. 3F). Compared
to MLV, Ba/F3 cells show a 100-fold block to M-PMV infection (Fig. 3F). Although M-PMV
infected cells showed normal levels of total viral DNA (GFP), a 60-fold reduction in the
formation of 2-LTR circles was observed, highlighting a block in nuclear entry (Fig. 3G).
Importantly, in comparison to MLV, we also observed a 6-fold reduction in histone H3
association with M-PMV total viral DNA (Fig. 3H). Together, our two independent approaches
highlight the nuclear entry requirement for efficient histone loading onto viral DNAs.
Loading of epigenetically modified histones onto viral DNAs is delayed compared to core
histones
To extend our above observation that retroviral DNAs undergo rapid association with
core histone H3 early during infection, we next examined the kinetics of epigenetically modified
histone loading onto retroviral DNAs. To do this, we made use of a previously established MLV
transcriptional silencing phenomenon in F9 mouse embryonic carcinoma cells. Flow cytometry
analysis of F9 cells transduced with MLV-GFP at various times post-infection is depicted in Fig.
4A. At 1 day post infection, modest GFP expression (13%) was observed. At 5 days, less than 1%
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of the cells were GFP+ (Fig. 4A). This data suggest that there is a lag to complete promoter
silencing. Based on this, we hypothesized that the delay in MLV silencing may be due to the
absence of repressive histone marks on the viral DNA. To test this, ChIP analysis from infected
cells were carried out using antibodies against histone H3K9 trimethylation (H3K9me3), a
marker of silenced chromatin, or acetyl-histone H3 (H3Ac), a marker of active chromatin,
followed by quantitative real-time PCR analysis using the indicated primers (Fig. 4B). As
controls, the specificity of these antibodies for ChIP was confirmed using positive control
primers specific for endogenous promoters Polrmt (H3K9me3) and GAPDH (H3Ac). During
early infection, there was very low enrichment of H3K9me3 marks on the viral DNA, consistent
with the modest number of GFP+ cells (Fig. 4B). In contrast, H3K9me3 became highly enriched
on the viral DNA by 6 days post infection (Fig. 4B), consistent with the complete silencing of
the viral promoter (Fig. 4A). Conversely, no enrichment of H3Ac marks on viral DNAs was
observed at all times (Fig. 5C). Together, these results suggest that the kinetics of modified
histone loading is slower than that of core histone loading, thereby contributing to the delay in
MLV silencing (Fig. 5A). As expected, deposition of repressive histone marks onto viral DNAs
is only seen in mouse embryonic cells, as reciprocal experiments in differentiated MEF cells
showed enrichment of H3Ac (Fig. 4E), but not H3K9me3 (Fig. 4D) marks on the viral DNA at 6
days post infection, consistent with high MLV gene expression in these cells.
Characterization of the expression of unintegrated MLV DNA in NIH-3T3 cells
Our findings that unintegrated MLV genomes are rapidly covered with core histones (Fig. 2D
and 2H) together with the observation that unintegrated MLV genomes show poor gene
expression (Fig. 2B) raised the possibility that unintegrated MLV DNA may lack specific
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chromatin modifications that prevent the recruitment of host transcriptional machinery. Histone
acetyltransferases and deacetylases are key components of the eukaryotic transcriptional
machinery that control the activation or silencing of cellular promoters. Acetylation of histone
H3 and H4 on lysine residues decrease the protein’s overall positive charge, thereby leading to
an open chromatin conformation conducive to transcription activation. In contrast, deacetylation
of histone H3 and H4 leads to chromatin condensation and transcriptional silencing. To test the
possibility that unintegrated MLV DNA may be deficient in histone acetylation, NIH-3T3 cells
were first infected with WT or D184A-IN GFP viruses for 24 h, followed by the addition of
Trichostatin A (TSA), a specific inhibitor of histone deacetylases (Fig. 5A). Interestingly,
following TSA treatment, GFP expression from cells infected with D184A-IN virus showed a 4-
fold increase in the percentage of GFP+ cells as well as a 3-fold increase in the mean
fluorescence intensity (Fig. 5B, upper panel). In contrast, TSA addition failed to increase GFP
expression in cells infected with WT-IN viruses (Fig. 5B, lower panel), suggesting that this drug
only affects expression from unintegrated viral DNAs. We next performed ChIP assays using
acetylated histone H3 antibodies before and after the addition of TSA in both WT and D184A-IN
mutant cells. Compared to mock treatment, TSA treatment of D184A-IN infected cells resulted
in a 4-fold increase in H3 acetylation of linear, but not 2-LTR circles (Fig. 5D). This suggests
that only linear, but not circular unintegrated viral DNA forms contribute to gene expression. In
contrast, exposure to TSA did not further increase the extent of histone H3 acetylation in WT-IN
infected cells (Fig. 5D), perhaps because fully integrated proviral DNA already exhibits high
enrichment in acetylated H3 marks. Together, these data suggest that gene expression from
unintegrated DNAs is hampered by the lack of necessary chromatin modifications that promotes
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RNA polymerase recruitment, and that the chromatin environment can be corrected by the
inhibition of histone deacetylases.
Characterization of the kinetics of nucleocapsid (NC) and capsid (CA) association with viral
DNA following reverse transcription
The NC proteins of retroviruses are small, highly basic nucleic acid binding proteins that directly
contacts the RNA genome in the mature virion. Apart from its structural role, proper NC is
indispensable for many steps of viral replication including viral assembly, genomic RNA
packaging, reverse transcription and integration (Mori et al, 2015). Here, we examined the fate of
viral DNAs following infection with respect to its association with NC. ChIP assays using MLV-
NC specific antibodies were performed at different times post infection in infected NIH-3T3
cells, followed by quantitative real time PCR to monitor the extent of NC association with total
viral DNA (GFP) and 2-LTR circles (Fig. 6A). Shown in Fig. 6A, highest association of NC with
the total viral DNA was observed 4 h post infection, followed by a rapid decrease to very low
levels by 48 h. Of note, no association between NC and 2-LTR circles was detected at all times
(Fig. 6A). Similar kinetics of association was observed between CA and total viral DNA (Fig.
6B). In each case, the specificity of the antibodies used for ChIP is confirmed by the lack of non-
specific binding to the cellular GAPDH gene, as well as using cells that were infected with heat
inactivated (HI) virus (Fig. 6A and 6B).
The timing of NC and CA dissociation from the total viral DNAs (Fig. 6A and 6B)
appears to coincide with the rapid accumulation of core histones (Fig. 1B and 1D). Since histone
loading of viral DNAs takes place in the nucleus (Fig. 3D and 3H), this raises the possibility that
upon nuclear entry, core histones may promote the dissociation of NC and CA from the viral
DNAs by direct competition for DNA binding. If this was the case, one would expect that
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preventing nuclear entry may result in the stabilization of NC and CA-DNA complexes. Indeed,
NC and CA ChIP analysis from aphidicolin treated NIH-3T3 cells showed a 3-fold increase in
NC and CA binding to total viral DNAs (Fig. 6C). This implies that histone and viral protein
binding to the viral DNA may be mutually exclusive.
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DISCUSSION
In this study, using MLV as a model for retroviruses, we described for the first time the
state of the viral DNAs following infection. By performing histone H3 ChIP analysis on murine
cells infected with MLV, we show that core histone H3 rapidly associates with total viral DNAs
following infection (Fig. 1B and 1D). Moreover, experiments using either integrase mutant MLV
or raltegravir treated cells demonstrate that histone loading of total viral DNAs occurs
independently of viral integration (Fig. 2D and 2H). Total viral DNA is composed of linear DNA,
a direct product of reverse transcription and a substrate for integration, circular DNA, a marker
of nuclear entry, and fully integrated proviral DNA. 2-LTR circles constitute 10-15% of total
viral DNAs (Fig. 1A and 1C), while the remaining is made up of mostly linear DNA, other forms
of circular DNA, and fully integrated proviral DNA. From this, we deduce that during early
infection, histone loading of linear DNA must account for a fraction of the total ChIP signal (Fig.
1B and 1D). Since linear DNA also serve as precursors to 2-LTR circles, one can envision
several potential mechanisms of histone loading onto 2-LTR circles, including (1) histone
loading onto linear DNA followed by circularization, (2) circularization followed by histone
loading, or (3) histone loading and DNA circularization occurring simultaneously. Our current
data cannot distinguish between these possible modes of histone loading.
The majority, if not all of the histone loading onto viral DNAs occurs in the nucleus. This
is supported by our observation that perturbation of viral nuclear entry, either by infecting
aphidicolin arrested cells with MLV (Fig. 3D), or by infecting mouse cells with M-PMV, a virus
previously shown to be defective in nuclear entry (Fig. 3H), all caused a marked reduction in the
extent of histone H3 association with total viral DNAs.
Using a previously characterized model of MLV promoter silencing in mouse embryonic
cells, we were able to directly measure the timing of epigenetically modified histone association
59
with viral DNAs. In F9 embryonic carcinoma cells, significant core histone loading occurred as
early as 12 h post infection (Fig. 1D). In contrast, modified histone loading, as measured by the
presence of H3K9 trimethylation (H3K9me3), became evident much later at 6 days post
infection (Fig. 4B). The delay in the appearance of modified histones on the viral DNAs
correlates with the delay in the complete transcriptional silencing of the MLV promoter (Fig.
4A). In the case of differentiated cells, where the MLV promoter is fully active, acetylated
histone H3 loading was also found to occur much later at 6 days compared to core histone
loading as early as 12 h (Fig. 4E). Together, these data suggest that the appearance of both
repressive and active histone marks occur after core histone loading, thereby causing a transient
delay in the host cell modulation of viral gene expression. In host cells, DNA replication during
S-phase is associated with rapid reassembly of nucleosomes and inheritance of parental histone
modifications (Zentner & Henikoff, 2013). This is achieved by histones around the parental
DNA strand acting as a template to direct histone modifications of the daughter DNA strand
(Zhu & Reinberg, 2011). Retroviral infection generates de novo foreign DNA that the cell has
never seen before, hence the delay in the appearance of histone marks may reflect the absence of
previously established epigenetic memory, and the timing required to establish it in the first
place.
Using a catalytically inactive IN mutant (D184A), we were able to study gene expression
derived from unintegrated viral DNAs. Under basal conditions, the expression level from
unintegrated viral DNAs was very low compared to integrated proviral DNAs (Fig. 5B),
consistent with previous reports (Nakajima et al, 2001; Poon & Chen, 2003). However, the
addition of HDAC inhibitor TSA following infection caused a marked increase in the expression
of unintegrated, but not integrated viral DNAs (Fig. 5B). The increase in expression from
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unintegrated DNAs appears to be a product of two mechanisms. First, the increase in gene
expression from unintegrated viral DNA was accompanied by an increase in the association of
acetylated histone H3, a marker of actively transcribed genes, with the viral promoter (Fig. 5D),
thus favoring the recruitment of the host transcriptional machinery. Interestingly, such H3Ac
enrichment was only observed on total viral DNA, but not 2-LTR circles, implying that the
enhanced gene expression may be a product of increased transcription from only linear DNA
templates and not circular forms (Fig. 5D). Secondly, TSA appears to increase the stability of
unintegrated, but not integrated DNA forms, thereby increasing the amount of viral template
available for transcription (Fig. 5C). Why this is the case is a topic for future investigations, one
possibility is that TSA treatment may prolong the half-life of unintegrated DNA forms by
reducing the activity of the DNA degradation machinery in the nucleus.
Using antibodies specific for the NC and CA proteins of MLV, we were able to follow
the kinetics of NC and CA dissociation from the viral DNA following infection (Fig. 6A and 6B).
High degree of NC and CA association with the viral DNA early during infection followed by
rapid decay coincides with the timing of histone loading, suggesting that the two events may be
mutually exclusive. Indeed, by blocking nuclear entry and preventing histone loading, we were
able to prolong the association of NC and CA with the viral DNAs (Fig. 6C). One possible
model is that following entry of viral DNAs into the nucleus, core histones may compete with
NC and CA for binding to the viral DNAs, leading to their disassembly (Fig. 7C).
The precise mechanism by which core histones are loaded onto retroviral DNAs remains
a mystery, and likely requires the activity of cellular histone chaperones. Since retroviral
infection generates newly synthesized DNA through reverse transcription, one may predict that
histone loading of viral DNA is analogous to histone loading of newly synthesized cellular DNA
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during DNA replication. For cellular DNA, the Caf1 complex is thought to mediated de novo
histone H3/H4 assembly of newly synthesized DNA during replication (Gaillard et al, 1996;
Kaufman et al, 1995). However, preliminary experiments using Caf1 knockdown cells showed
no impairment in the extent of histone loading onto viral DNAs (data not shown), highlighting
the likely involvement of other chaperones, a topic of future studies. Equally interesting is the
question of whether viral encoded proteins play a role in facilitating histone loading onto its own
DNA.
Future studies will be aimed at elucidating the effect of histone loading on the retroviral
replication potential. For instance, does histone loading protects linear DNA from degradation?
Does histone loading of linear DNA promote efficient integration or integration site selection?
Answering these questions will require an understanding of the cellular machinery responsible
for histone loading onto viral DNAs.
62
MATERIALS AND METHODS
Cell lines and antibodies
Cell lines including human embryonic kidney (HEK)-293 (CRL-1573), F9 embryonic carcinoma
(CRL-1720), Mouse embryonic fibroblasts (MEF) (SCRC-1008) and NIH-3T3 (CRL-1658) cells
were purchased from American Type Culture Collection. All cells were cultured in Dulbecco’s
modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum
(FBS), 2 mM glutamine, 1000 U/mL penicillin and 100 mg/mL streptomycin. Ba/F3 cells were
cultured in RPMI medium supplemented with 10% heat-inactivated FBS and 1.6 ng/mL of
recombinant IL-3 (R&D Systems). All cells were maintained in a 37°C incubator with 5% CO2.
Antibodies used in this study include: Anti-Histone H3 (ab1791, Abcam), Anti-Histone H3 tri-
methyl K9 (ab8898, Abcam), Anti-Acetyl-Histone H3 (06-599, Millipore), anti-MLV NC
(National Cancer Institute serum 79S-), anti-MLV CA (National Cancer Institute serum 79S-804)
and rabbit IgG control antibody (sc-2027, Santa Cruz).
Plasmids
pCMV-intron expresses wild-type gag and pol from NB-tropic MLV (Soneoka et al, 1995).
pCMV-intron D184A-IN is an integrase dead mutant of pCMV-intron created by site directed
mutagenesis. pMD.G expresses the vesicular stomatitis virus (VSV) envelope glycoprotein.
pNCA-GFP is a replication-defective single-round MLV vector described previously (Ooi et al,
2010). pSARM-EGFP is a replication-defective single-round M-PMV vector in which the env
gene has been replaced with EGFP as described (Newman et al, 2006).
Retroviral transduction assay and flow cytometry analysis
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NB-tropic MLV-GFP reporter viruses were produced by 293T cell transfection with 8 g of
pNCA-GFP, 4 g of pCMV-intron, and 4 g of pMD.G DNAs using Polyethylenimine (PEI).
Similarly, M-PMV-GFP reporter viruses were produced by transfection of 293T cells with 12 g
of pSARM-EGFP and 4 g of pMD.G. All reporter viruses were harvested 48 h later, filtered
(0.45 m), DNase treated for 1 h (Turbo DNase, Thermo Scientific), and used directly for
transduction assays as described previously (Wang & Goff, 2015). For some experiments, 10 M
of raltegravir (Santa Cruz Biotechnology) was added to the culture medium for the entire
duration of infection. Forty-eight hours post-infection, cells were trypsinized, diluted using flow
cytometry buffer (PBS with 1% BSA), and subjected to flow cytometry using automated cell
analyzer (LSRII, BD Bioscience).
Chromatin immunoprecipitation
5×106 cells were crosslinked with 1% formaldehyde at room temperature for 10 min, followed
by quenching with 0.125M glycine for 5 min. Cells were lysed in 0.5 mL of ChIP lysis buffer
(50mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) containing Protease inhibitor cocktail (Roche)
and sonicated to produce an average fragment size of 200–800 base pairs. Each
immunoprecipitation was performed by incubating 30 g of sonicated chromatin together with 4
g of respective antibodies in ChIP dilution buffer (10 mM Tris-HCl pH 8.0, 1% Triton X-100,
0.1% SDS, 150 mM NaCl, 2 mM EDTA) overnight at 4°C. Next day, 25 μl of Protein A/G
Dynabeads (Life Technologies) were added for an additional 4 h. Captured antibody-antigen
complexes were washed 2 times each in ChIP low salt buffer (20 mM Tris-HCl pH 8.0, 1%
Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM EDTA), ChIP high salt buffer (20 mM Tris-HCl
pH 8.0, 1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA), ChIP LiCl buffer (10 mM
64
Tris-HCl pH 8.0, 1% NP-40, 250 mM LiCl, 1 mM EDTA), and TE buffer (10 mM Tris-HCl pH
8.0, 1 mM EDTA). DNA was eluted from beads in 200 μl elution buffer (TE buffer containing 1%
SDS, 100 mM NaCl, 5mM DTT), reverse crosslinked (65°C overnight), treated with RNase A
(37°C, 1 h) and Proteinase K (37°C, 2 h), and purified using QIAquick PCR purification kit
according to the manufacturer’s instructions (Qiagen). Quantitative real-time PCRs were
performed with indicated primers. The number of copies of each PCR product in both input and
immunoprecipitated DNA was first determined using plasmid derived standard curves. Relative
enrichment is shown as percent of input DNA calculated by dividing the number of copies from
immunoprecipitated DNA by input DNA and multiplying it by 100%.
Quantitative real-time PCR analysis of viral replication intermediates
At various time points following infection, cells were washed with PBS, and total DNA was
isolated using Qiagen DNeasy kit according to the manufacturer’s instructions. For analysis of
viral replication intermediates, 75 ng of total DNA was mixed with SYBR Green PCR master
mix (Roche) containing 15 pmol of indicated primers (see Supplemental table 1). PCRs were
performed in 96-well plates using 7900 Fast Real-Time PCR system (Applied Biosystems) with
the following reaction conditions: 10 min at 95°C, followed by 45 cycles of 30 s at 95°C, 30 s at
60°C and 30 s at 72°C. For all reactions, the number of copies of each PCR product was
determined using plasmid derived standard curves.
Aphidicolin treatment and cell cycle analysis of DNA content
NIH-3T3 cells were treated with aphidicolin (Sigma, 2 g/mL) for 24 h prior to infection.
Efficiency of aphidicolin treatment was confirmed by analyzing DNA content. Cells were
65
stained with Hoechst 33342 (Invitrogen) at 1 g/mL in cell culture medium for 30 min at 37°C,
followed by flow cytometry using automated cell analyzer (LSRII, BD Bioscience).
66
ACKNOWLEDGEMENTS
This work was supported by NCI grant R01 CA 30488 from the National Cancer Institute. S.P.G.
is an investigator of the Howard Hughes Medical Institute.
AUTHOR CONTRIBUTIONS
GZW and SPG designed and supervised the study. GZW and YW conducted all the experiments.
GZW and SPG analyzed the data and wrote the manuscript.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
67
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FIGURE LEGENDS
FIGURE 1. Core histone loading onto retroviral DNAs occur rapidly following infection
(A) MEF cells were infected with VSV-G pseudotyped MLV-GFP reporter virus. To control for
potential plasmid DNA carry over in the viral supernatant, heat inactivated (HI) virus were used
in parallel. Total DNA from infected cells was isolated at indicated time points, followed by real
time quantitative PCR using primers targeting GFP (total viral DNA) and 2-LTR circles.
Absolute copy number is calculated based on standard curves generated using plasmid DNA.
Results shown are means ± SDs from two independent experiments performed in duplicates.
(B) Histone H3 chromatin immunoprecipitation (ChIP) analysis of chromatin harvested at
indicated time points following MLV-GFP infection of MEF cells. ChIP data is presented as %
of input DNA, calculated by dividing the ChIP copy number for each gene target by the copy
number from input DNA and multiplying by 100%. Results shown are means ± SDs from two
independent experiments performed in duplicates. ND denotes not determined.
(C) ChIP analysis as outlined in (B) using rabbit IgG control antibody.
(D) Same experiment as outlined in (A) using F9 embryonic carcinoma cells.
(E) Same experiment as outlined in (B) using F9 embryonic carcinoma cells.
(F) Same experiment as outlined in (C) using F9 embryonic carcinoma cells.
FIGURE 2. Histone loading of retroviral DNAs occurs independently of viral integration
(A) Schematic of experimental setup.
(B) Flow cytometry analysis of NIH-3T3 cells infected with VSV-G pseudotyped wild-type (WT)
or integrase dead mutant (D184A) MLV-GFP reporter viruses. This is 2 days post infection. Y-
axis shows side scatter (SSC), X-axis shows GFP intensity. One representative experiment of
three independent experiments is shown.
(C) NIH-3T3 cells were infected with VSV-G pseudotyped wild-type (WT) or integrase dead
mutant (D184A) MLV-GFP reporter viruses. Total DNA from infected cells was isolated at
indicated time points post infection and number of copies of viral replication intermediates was
determined as described above. Results shown are means ± SDs from two independent
experiments performed in duplicates.
(D) Histone H3 ChIP analysis of chromatin harvested at indicated time points following MLV-
GFP infection of NIH-3T3 cells. ChIP data is presented as % of input DNA as described above.
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Results shown are means ± SDs from two independent experiments performed in duplicates. ND
denotes not determined.
(E) Schematic of experimental setup.
(F) Flow cytometry analysis of MEF cells infected with VSV-G pseudotyped WT MLV-GFP
reporter viruses in the presence or absence of raltegravir (Ral). This is 2 days post infection. Y-
axis shows side scatter (SSC), X-axis shows GFP intensity. One representative experiment of
three independent experiments is shown.
(G) MEF cells were infected with VSV-G pseudotyped wild-type (WT) MLV-GFP reporter
viruses in the presence or absence of raltegravir (Ral). Total DNA from infected cells was
isolated at indicated time points post infection and the number of copies of viral replication
intermediates was determined as described above. Results shown are means ± SDs from two
independent experiments performed in duplicates.
(H) Histone H3 ChIP analysis of chromatin harvested at indicated time points following MLV-
GFP infection of MEF cells treated with or without raltegravir (Ral). ChIP data is presented as %
of input DNA as described above. Results shown are means ± SDs from two independent
experiments performed in duplicates. ND denotes not determined.
FIGURE 3. Histone loading of retroviral DNAs requires viral nuclear entry
(A) Schematic of experimental setup.
(B) Cell cycle analysis of NIH-3T3 cells treated with or without DNA polymerase inhibitor
aphidicolin for 24 h prior to infection. Y-axis shows cell count, X-axis shows DNA content
measured by Hoechst staining. One representative experiment of three independent experiments
is shown.
(C) NIH-3T3 cells pretreated with or without aphidicolin (Aph) to induce cell cycle arrest were
infected with VSV-G pseudotyped WT MLV-GFP reporter virus. Total DNA from infected cells
was isolated 24 h post infection and the levels of viral replication intermediates were determined
as described above. Results shown are means ± SDs from two independent experiments
performed in duplicates.
(D) Histone H3 ChIP analysis of chromatin harvested from NIH-3T3 cells outlined in (C) 24 h
post infection. ChIP data is presented as % of input DNA as described above. Results shown are
means ± SDs from two independent experiments performed in duplicates. ND denotes not
determined.
(E) Schematic of experimental setup.
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(F) Flow cytometry analysis of Ba/F3 cells infected with VSV-G pseudotyped single-round
MLV or M-PMV-GFP reporter viruses. This is 2 days post infection. Y-axis shows side scatter
(SSC), X-axis shows GFP intensity. One representative experiment of three independent
experiments is shown.
(G) Level of viral replication intermediates from Ba/F3 cells infected with MLV or M-PMV-
GFP reporter viruses. Total DNA from infected cells was isolated at indicated time points post
infection and number of copies of viral replication intermediates was determined as described
above. Results shown are means ± SDs from two independent experiments performed in
duplicates.
(H) Histone H3 ChIP analysis of chromatin harvested from infected Ba/F3 cells outlined in (G).
ChIP data is presented as % of input DNA as described above. Results shown are means ± SDs
from two independent experiments performed in duplicates. ND denotes not determined.
FIGURE 4. Loading of epigenetically modified histones onto viral DNAs is delayed compared
to core histones
(A) Flow cytometry analysis of F9 cells infected with VSV-G pseudotyped WT MLV-GFP
reporter viruses at the indicated time points. Y-axis shows side scatter (SSC), X-axis shows GFP
intensity. One representative experiment of three independent experiments is shown.
(B) H3K9 trimethylation (H3K9me3) ChIP analysis of infected F9 cells at the indicated time
points. ChIP data is presented as % of input DNA as described above. Results shown are means
± SDs from two independent experiments performed in duplicates. Mitochondrial DNA
polymerase (positive control gene), GAPDH (negative control gene). ND denotes not determined.
(C) Acetyl-histone H3 (H3Ac) ChIP analysis of infected F9 cells at the indicated time points.
ChIP data is presented as % of input DNA as described above. Results shown are means ± SDs
from two independent experiments performed in duplicates. Mitochondrial DNA polymerase
(negative control gene), GAPDH (positive control gene). ND denotes not determined.
(D) Similar experiment as (B) performed in MEF cells.
(E) Similar experiment as (C) performed in MEF cells.
FIGURE 5. Characterization of the expression of unintegrated MLV DNA in NIH-3T3 cells
(A) Schematic of experimental setup.
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(B) Flow cytometry analysis of NIH-3T3 cells infected with VSV-G pseudotyped wild-type (WT)
or integrase dead mutant (D184A) MLV-GFP reporter viruses in the presence or absence of TSA
treatment. This is 2 days post TSA treatment. Y-axis shows side scatter (SSC), X-axis shows
GFP intensity. One representative experiment of three independent experiments is shown.
(C) NIH-3T3 cells infected with VSV-G pseudotyped WT or integrase dead mutant (D184A)
MLV-GFP reporter virus and each treated with or without TSA. Total DNA from infected cells
was isolated at indicated time points and levels of viral replication intermediates were
determined as described above. Results shown are means ± SDs from two independent
experiments performed in duplicates.
(D) H3Ac ChIP analysis of infected NIH-3T3 cells treated with or without TSA. ChIP data is
presented as % of input DNA as described above. Results shown are means ± SEMs from three
independent experiments performed in duplicates. Mitochondrial DNA polymerase (negative
control gene), GAPDH (positive control gene).
FIGURE 6. Characterization of the kinetics of nucleocapsid (NC) and capsid (CA) association
with viral DNA following reverse transcription
(A) MLV-NC ChIP analysis of chromatin harvested at indicated time points following MLV-
GFP infection of NIH-3T3 cells. ChIP data is presented as % of input DNA as described above.
Results shown are means ± SDs from two independent experiments performed in duplicates.
(B) Similar experiment as (A) using MLV-CA antibodies for ChIP.
(C) MLV-NC and CA ChIP analysis of chromatin harvested from infected, aphidicolin (Aph)
treated NIH-3T3 cells. ChIP data is presented as % of input DNA as described above. Results
shown are means ± SDs from two independent experiments performed in duplicates.
FIGURE 7. Model of the states of retroviral DNA following infection
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CHAPTER 6 - DISCUSSION
The data presented in the previous chapters encompasses various aspects of the retroviral life
cycle during early phases of infection. In the following section, I will individually discuss the
implications of our findings and suggest some future investigations.
EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney
murine leukemia virus in embryonic cells
I began my graduate work studying the phenomenon of MLV silencing in mouse embryonic cells
(Chapter 2). The ability of mouse embryonic cells to silence the MLV promoter lies in its
expression of the sequence specific KRAB-ZFP ZFP809. ZFP809, through its interaction with
the PBSpro of MLV, serves to coordinate the assembly of macromolecular silencing complexes
containing TRIM28, ESET, HP1 and others, thereby establishing of a transcriptionally inactive
provirus. As a continuation of work done by Dr. Daniel Wolf in the lab, we identified a novel
ZFP809 interacting protein, EBP1, and showed its involvement in facilitating ZFP809/TRIM28
dependent MLV silencing in mouse embryonic cells. There was no previous history of an
involvement of EBP1 in ES-cell specific silencing. EBP1 is a known transcriptional repressor of
E2F1 and androgen receptor regulated promoters (Ghosh et al, 2013; Zhang et al, 2005a; Zhang
et al, 2002; Zhang et al, 2005b; Zhang et al, 2003) in breast and prostate cancer cell lines.
Interestingly, our observation of the interaction between EBP1 and TRIM28 coincides with
previous findings showing the involvement of both proteins at the E2F target promoter (Wang et
al, 2007). Therefore, it is conceivable that for other cellular promoters, specific DNA binding
proteins, acting in a manner similar ZFP809, may recruit both EBP1 and TRIM28 to the target
promoter and modulate gene expression epigenetically. Given that both EBP1 and TRIM28 are
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ubiquitously expressed and could regulate target genes in many cell types, we postulate that cell
specific promoter regulation will be determined by cell specific zinc finger proteins, which in the
case of embryonic cells, is ZFP809.
Of note, EBP1 has been implicated in the biology of RNA viruses in other settings.
Honda et al identified EBP1 as a novel binding partner for the PB1 subunit of influenza virus
RNA polymerase and showed that overexpression of EBP1 blocked the transcription of influenza
virus and led to reduced viral titers (Honda et al, 2007). In another study, it was reported that
infection of Vero cells by rinderpest virus led to the downregulation of EBP1 expression, and
that overexpression of EBP1 led to the inhibition of rinderpest virus transcription (Gopinath et al,
2010). EBP1’s role in antagonizing retroviral infection in embryonic cells expands the repertoire
of RNA viruses sensitive to EBP1 regulation. Preliminary tests suggest that cellular EBP1
expression is not altered by retroviral infection (data not shown), and that the course of viral
DNA synthesis by reverse transcriptase was not affected.
Mouse embryonic cells suppress the expression of both incoming exogenous retroviruses
as well as endogenous retroelements. The loss of either ZFP809 (Wolf et al, 2015), TRIM28
(Rowe et al, 2010) or SETDB1 (Matsui et al, 2010) in mouse ES cells led to robust activation of
endogenous retroviruses, as well as failure to block new retroviral infections. In F9 EC cells,
depletion of EBP1 did not result in the upregulation of various endogenous retroelements (data
not shown). Therefore, we suggest that although EBP1 is required for the silencing of newly
integrated provirus, it may be dispensable for the silencing of endogenous retroviral elements.
Such division of labor has also been observed in the case of the histone methyltransferase G9a
(Leung et al, 2011).
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In summary, our study revealed EBP1 to be a novel component of the PBS-dependent
silencing machinery in mouse embryonic cells. It should be noted that retroviral silencing is
mediated through both PBS-dependent, as well as PBS-independent pathways involving the host
transcription factor Yin Yang 1 (Schlesinger et al, 2013), and further studies will be necessary to
address the potential crosstalk between these two arms. It will also be of interest to determine
whether TRIM28 in concert with other KRAB-ZFPs also requires EBP1 to mediate silencing in
other settings.
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Regulation of Yin Yang 1 by Tyrosine Phosphorylation
As an extension of YY1’s role in MLV silencing, I became interested in understanding
how YY1 activity is regulated through post-translational modifications. In chapter 3, I showed
that YY1 is a target of tyrosine phosphorylation across many cell types. Using a combination of
kinase inhibitors, kinase overexpression and kinase knockout studies, we arrived at the
conclusion that YY1 phosphorylation is likely mediated by multiple Src family kinases. The nine
Src family kinases exhibit different tissue expression patterns. For example, c-Src, Fyn, and Yes
are ubiquitously expressed (Stein et al, 1994), while Lyn shows preferential expression in
hematopoietic cells (Hibbs et al, 1995). Given the conservation of YY1 phosphorylation across
different cell types, we postulate that depending on the Src kinase signature within a cell,
different cell types may utilize different members of the kinase family for phosphorylating YY1.
Thus, this mechanism of YY1 regulation may be functioning in a wide range of tissues and
settings.
In Lyn-overexpressing cells, overall YY1 phosphorylation was greatly reduced by single
mutations at either tyrosine 8, 254 or 383, suggesting that these three sites are preferentially
phosphorylated by Lyn, and that phosphorylation of these sites may occur in a cooperative
manner, since mutations at each site greatly reduced the likelihood that the other sites become
phosphorylated in vivo. Phosphorylation of bacterially purified Y383F YY1 using recombinant
Lyn was also significantly reduced in vitro. However, in both Src and Yes overexpressing cells,
YY1 was preferentially phosphorylated on different residues, suggesting that the sites of YY1
phosphorylation in a given cell type may depend on the expression pattern of Src family kinases
within that cell. Importantly, it appears that tyrosine 383 is a target of all three kinases.
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To examine the functional importance of YY1 phosphorylation sites uncovered by our
mutagenesis analysis, we tested the effect of introducing phosphomimetic and
nonphosphorylatable mutations on the ability of YY1 to regulate gene expression driven by both
retroviral and cellular promoters. Our observation that overexpression of Y383E, but not Y383F,
affected the ability of YY1 to either transactivate the HTLV-1 promoter, or repress the MLV or
endogenous c-fos promoter highlighted the functional importance of this tyrosine residue.
YY1 contains four C2H2 zinc finger domains, each of which is comprised of conserved
cysteines, histidines and hydrophobic residues folding to form a structure surrounding a
central zinc ion (Houbaviy et al, 1996). Y383 is located at the very start of the fourth zinc finger,
before the first cysteine of the CCHH cluster chelating the zinc, and is not the highly conserved
bulky hydrophobic residue in the center of the zinc finger. A closer inspection of the co-crystal
structure of YY1 bound to the AAV P5 promoter initiator element (Houbaviy et al, 1996) reveals
that tyrosine 383, together with valine 384 and critical cysteine 385 participate in the formation
of the first -sheet of the fourth zinc finger. Hence, one would predict that the addition of a
phosphate at this position may perturb the first -sheet, thus interfering with zinc and/or nucleic
acid binding. We cannot rule out the possibility that phosphorylation induced changes in protein
folding may also affect YY1’s affinity for other transcriptional cofactors such as CTCF
(Donohoe et al, 2007) and Smads (Kurisaki et al, 2003).
The importance of Y383 is supported by biochemical assays showing reduced DNA and
RNA binding by the Y383E mutant. Apart from tyrosine 383, it is surprising that
phosphomimetic mutations at the other five tyrosines failed to affect YY1 function in the context
of HTLV-1 transactivation. For example, tyrosine 185 lies in close proximity to serine 180 and
serine 184, sites previously shown to be a target of Aurora B kinase, leading to changes in DNA
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binding and YY1 acetylation (Kassardjian et al, 2012). In spite of this proximity, the Y185E and
Y185F mutants maintained full transactivation activity on the HTLV-1 promoter. Therefore, the
simplest explanation is that phosphorylation of the other five tyrosines is not important for
YY1’s activation function.
Inactivation of gene-specific transcription factors often affects control of cell division and
mitosis. One mechanism of transcription factor inactivation during the cell cycle involves
serine/threonine phosphorylation of the consensus TGEKP zinc finger linker sequence in C2H2
transcription factors such as Sp1, Ikaros (Dovat et al, 2002) and YY1 (Rizkallah et al, 2011a;
Rizkallah & Hurt, 2009), leading to reduced DNA binding., YY1 activity may also be similarly
controlled during the cell cycle by phosphorylation of tyrosine 383. To our knowledge,
regulation of a C2H2 transcription factor through tyrosine phosphorylation of its zinc finger has
not been documented. In YY1, tyrosine 383 makes up part of the consensus Y-X-C motif that is
also found at the start of many other C2H2 transcription factors (Brayer & Segal, 2008), thereby
raising that possibility that other C2H2 family members may be regulated in a similar manner.
YY1 is a stable, constitutive and widely expressed protein across many cell types (Austen
et al, 1997). This makes regulation of YY1 through post-translational modifications an attractive
mean of controlling its activity. The levels of tyrosine phosphorylated YY1 is low under basal
conditions, but becomes readily detectable upon the inhibition of endogenous tyrosine
phosphatases or the activation of cytosolic tyrosine kinases via EGFR signaling. Given that
protein phosphorylation is dictated by the balance between kinase and phosphatase activities, our
data suggests that the majority of YY1 is unphosphorylated and available for DNA binding under
steady-state conditions, leading to persistent activation or repression of YY1 responsive genes.
However, the presence of extracellular stimuli such as EGF allows for dynamic changes in the
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DNA binding capacity of YY1 through tyrosine phosphorylation, thereby permitting temporal
fine-tuning of YY1-responsive promoters.
Under steady-state conditions, YY1 is predominantly a nuclear protein with a minor
cytoplasmic presence (Austen et al, 1997). Cellular fractionation experiments using pervanadate-
treated Jurkat cells showed high levels of tyrosine phosphorylated YY1 in the nucleus and trace
amounts in the cytoplasm. This is consistent with emerging evidence showing that beside
cytoplasmic localization, a fraction of the Lyn (Radha et al, 1996), Src, Fyn, and Yes kinases
(Takahashi et al, 2009) is present in the nucleus. Nevertheless, the interplay between YY1
phosphorylation and cellular localization is a topic of future studies.
In summary, our work has revealed a novel means of regulating YY1 function through
tyrosine phosphorylation. Further studies will be necessary to address the specific tyrosine
phosphatase(s) responsible for YY1 dephosphorylation, as well as the localization and dynamics
of tyrosine phosphorylated YY1 in vivo. These studies will provide us with a better
understanding of the complex interplay between tyrosine kinases and phosphatases in regulating
YY1 function.
88
Postentry restriction of Mason-Pfizer monkey virus in mouse cells
My next project revolved around an interesting observation that mouse cells are resistant
to infection by M-PMV, a prototypical simian betaretrovirus. As shown in chapter 4, the block is
very potent (50-100 fold) and resistance to M-PMV appears to be widespread among many cell
types. Moreover, the observation that mouse cells blocked M-PMV reporter viruses pseudotyped
with various envelope proteins suggests that the block occurs post-entry. Moreover, the process
of reverse transcription, as measured by the levels of minus-strand strong-stop DNA and the
reporter GFP DNA, occurred normally in all the mouse cell lines tested. The levels of 2-LTR
circles, however, were drastically reduced, suggesting a significant block at the time of nuclear
entry. This conclusion is further supported by diminished levels of integrated proviral DNA
remaining in mouse cells after long-term passage. It is not known if infection of permissive cells
by M-PMV requires cell division as is the case for MLVs, but the cells used in all our
experiments were dividing, ensuring that this is not the basis for the block. Given that the defect
in viral circular DNA formation partially coincides with the timing of Fv1 restriction, we
hypothesized that the Fv1 alleles present in the various mouse lines might account for the block
in M-PMV infection. But overexpression of the Fv1n or Fv1
b alleles in human cells failed to
induce any resistance to M-PMV. This observation was consistent with the inability of M-PMV
to transduce mouse cells harboring different Fv1 alleles, making Fv1 an unlikely candidate for
M-PMV restriction. Interestingly, it has been reported that Fv1 also failed to block primate
lentiviruses (Hatziioannou et al, 2004). Of note, although our data suggest a major defect at the
approximate time of nuclear entry, we cannot exclude the possibility that additional blocks at the
level of integration per se may also exist in mouse cells.
89
The inability of M-PMV to replicate in mouse cells may be due to either the absence or
incompatibility of critical host factors, or to the presence of active viral restriction factors
exemplified by Fv1. Infection of heterokaryons formed by the fusion between permissive and
non-permissive cells can be used to distinguish between the two possibilities. In preliminary tests,
we found that heterokaryons formed by the fusion of HeLa and NIH-3T3 cells remained resistant
to M-PMV infection (data not shown), implying that mouse cells may contain a novel dominant
restriction factor with Fv1-like activity. We cannot rule out the possibility, however, that the
fusion process itself is causing virus resistance and that the heterokaryons are resistant for other
reasons. Recently, the effects of human and mouse TRIM proteins on the early and late events of
HIV and MLV life cycle identified numerous family members with antiviral activities (Uchil et
al, 2008). With that in mind, it is possible that a ubiquitously expressed mouse-specific TRIM
protein is responsible for the M-PMV resistance. Preliminary tests of a panel of mouse TRIMs
expressed in HeLa cells did not reveal any TRIM capable of blocking M-PMV. Identification of
the putative dominant mouse restriction factor, if present, remains a subject for future
investigations. Finally, although mouse cells are non-permissive to M-PMV transduction, we
note that they are susceptible to infection by other betaretroviruses such as mouse mammary
tumor virus (MMTV). It remains to be determined whether this is due to MMTV’s ability to
escape host restriction, or the presence of compatible co-factors necessary for infection in mouse
cells.
90
Histones are rapidly loaded onto unintegrated retroviral DNAs soon after nuclear entry
Finally, the last project examines the question of what is the “state” of the viral DNAs
following infection. Given that reverse transcription generates de novo foreign DNAs in the host
cell, this raises the question of what is the histone state of these retroviral DNAs following
infection. Are retroviral DNAs a target of histone loading? If so, when, where and on what forms
of viral DNA does this occur? Following reverse transcription, do retroviral DNAs remain
associated with their cognate structural proteins such as NC and CA? When and where does NC
and CA dissociate from the viral DNA? Finally, given that unintegrated viral DNAs contribute
very little to viral gene expression in the infected cell, why is this so?
By performing histone H3 ChIP analysis on murine cells infected with MLV, we show
that core histone H3 rapidly associates with total viral DNAs following infection. Moreover,
experiments using either integrase mutant MLV or raltegravir treated cells demonstrate that
histone loading of total viral DNAs occurs independently of viral integration. Total viral DNA is
composed of linear DNA, a direct product of reverse transcription and a substrate for integration,
circular DNA, a marker of nuclear entry, and fully integrated proviral DNA. 2-LTR circles
constitute 10-15% of total viral DNAs, while the remaining is made up of mostly linear DNA,
other forms of circular DNA, and fully integrated proviral DNA. From this, we deduce that
during early infection, histone loading of linear DNA must account for a fraction of the total
ChIP signal. Since linear DNA also serve as precursors to 2-LTR circles, one can envision
several potential mechanisms of histone loading onto 2-LTR circles, including (1) histone
loading onto linear DNA followed by circularization, (2) circularization followed by histone
loading, or (3) histone loading and DNA circularization occurring simultaneously. Our current
data cannot distinguish between these possible modes of histone loading.
91
The majority, if not all of the histone loading onto viral DNAs occurs in the nucleus. This
is supported by our observation that perturbation of viral nuclear entry, either by infecting
aphidicolin arrested cells with MLV, or by infecting mouse cells with M-PMV, a virus
previously shown to be defective in nuclear entry, all caused a marked reduction in the extent of
histone H3 association with total viral DNAs.
Using a previously characterized model of MLV promoter silencing in mouse embryonic
cells, we were able to directly measure the timing of epigenetically modified histone association
with viral DNAs. In F9 embryonic carcinoma cells, significant core histone loading occurred as
early as 12 h post infection. In contrast, modified histone loading, as measured by the presence
of H3K9 trimethylation (H3K9me3), became evident much later at 6 days post infection. The
delay in the appearance of modified histones on the viral DNAs correlates with the delay in the
complete transcriptional silencing of the MLV promoter. In the case of differentiated cells,
where the MLV promoter is fully active, acetylated histone H3 loading was also found to occur
much later at 6 days compared to core histone loading as early as 12 h. Together, these data
suggest that the appearance of both repressive and active histone marks occur after core histone
loading, thereby causing a transient delay in the host cell modulation of viral gene expression. In
host cells, DNA replication during S-phase is associated with rapid reassembly of nucleosomes
and inheritance of parental histone modifications (Zentner & Henikoff, 2013). This is achieved
by histones around the parental DNA strand acting as a template to direct histone modifications
of the daughter DNA strand (Zhu & Reinberg, 2011). Retroviral infection generates de novo
foreign DNA that the cell has never seen before, hence the delay in the appearance of histone
marks may reflect the absence of previously established epigenetic memory, and the timing
required to establish it in the first place.
92
Using a catalytically inactive IN mutant (D184A), we were able to study gene expression
derived from unintegrated viral DNAs. Under basal conditions, the expression level from
unintegrated viral DNAs was very low compared to integrated proviral DNAs, consistent with
previous reports (Nakajima et al, 2001; Poon & Chen, 2003). However, the addition of HDAC
inhibitor TSA following infection caused a marked increase in the expression of unintegrated,
but not integrated viral DNAs. The increase in expression from unintegrated DNAs appears to be
a product of two mechanisms. First, the increase in gene expression from unintegrated viral DNA
was accompanied by an increase in the association of acetylated histone H3, a marker of actively
transcribed genes, with the viral promoter, thus favoring the recruitment of the host
transcriptional machinery. Interestingly, such H3Ac enrichment was only observed on total viral
DNA, but not 2-LTR circles, implying that the enhanced gene expression may be a product of
increased transcription from only linear DNA templates and not circular forms. Secondly, TSA
appears to increase the stability of unintegrated, but not integrated DNA forms, thereby
increasing the amount of viral template available for transcription. Why this is the case is a topic
for future investigations, one possibility is that TSA treatment may prolong the half-life of
unintegrated DNA forms by reducing the activity of the DNA degradation machinery in the
nucleus.
Using antibodies specific for the NC and CA proteins of MLV, we were able to follow
the kinetics of NC and CA dissociation from the viral DNA following infection. High degree of
NC and CA association with the viral DNA early during infection followed by rapid decay
coincides with the timing of histone loading, suggesting that the two events may be mutually
exclusive. Indeed, by blocking nuclear entry and preventing histone loading, we were able to
prolong the association of NC and CA with the viral DNAs. One possible model is that following
93
entry of viral DNAs into the nucleus, core histones may compete with NC and CA for binding to
the viral DNAs, leading to their disassembly.
The precise mechanism by which core histones are loaded onto retroviral DNAs remains
a mystery, and likely requires the activity of cellular histone chaperones. Since retroviral
infection generates newly synthesized DNA through reverse transcription, one may predict that
histone loading of viral DNA is analogous to histone loading of newly synthesized cellular DNA
during DNA replication. For cellular DNA, the Caf1 complex is thought to mediated de novo
histone H3/H4 assembly of newly synthesized DNA during replication (Gaillard et al, 1996;
Kaufman et al, 1995). However, preliminary experiments using Caf1 knockdown cells showed
no impairment in the extent of histone loading onto viral DNAs (data not shown), highlighting
the likely involvement of other chaperones, a topic of future studies. Equally interesting is the
question of whether viral encoded proteins play a role in facilitating histone loading onto its own
DNA.
Future studies will be aimed at elucidating the effect of histone loading on the retroviral
replication potential. For instance, does histone loading protects linear DNA from degradation?
Does histone loading of linear DNA promote efficient integration or integration site selection?
Answering these questions will require an understanding of the cellular machinery responsible
for histone loading onto viral DNAs.
94
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