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I INTRODUCTION
1. The Baculovirus Expression Vector System
2. Baculovirus Infection Process
3. Genes Involved in Viral Transcription and Replication
4. Baculovirus Host Range and Choice of Cells
5. Regulation of the Polyhedrin Gene Promoter
6. Aims and Objectives
Introduction 2
1. The Baculovirus Expression Vector System ·
The Baculovirus Expression Vector System (BEVS) has emerged as the
system of choice for the expression of foreign genes (for reviews [see
Luckow, 1991; Jarvis and Summers, 1992; O'Reilly et a/., 1992; Sridhar
et a/., 1994). Baculoviruses are insect viruses that infect mainly
· lepidopteran larvae. The Autographa californica nuclear polyhedrosis
virus (AcNPV) is most commonly used for expression work. The basic
biology centered around polyhedrin (polh ) gene expression during the
infection process underlies the utility of these viruses as expression
vectors (Miller, 1981 ). The polh gene of this virus, non-essential for the
replication of the virus in cell culture (Smith et a/., 1983), is expressed
to enormous levels during the late phases of infection. Therefore, in
most expression vectors, the poI h gene is replaced with the
heterologous gene which. is transcribed under the control of the
powerful polh promoter.
There are several features of the baculovirus system that are
particularly advantageous. Because an eukaryotic environment is being
used for protein production, biologically active proteins can be easily
obtained. Post-translational modifications like signal cleavage,
proteolytic cleavage, N-glycosylation, 0-glycosylation, acylation,
· amidation, phosphorylation, prenylation, and carboxyrnethylation can be
easily carried out by insect cells (O'Reilly et a/., 1992). Recombinant
proteins are targeted to their natural locations in the cell while
proteins containing signal peptides are recognized and cleaved in the
right fashion. The efficiency of secretion can be further enhanced by. __ _
fusing the honey bee melittin signal peptide (Tessier et a/., 1991 ).
Hetero- and homo-oligomeric assemblies have been demonstrated for a
wide variety of proteins in cells infected with baculoviruses. Using a
---
Introduction 3
baculovirus quadruple expression vector, four blue-tongue virus
proteins have been co-expressed and shown to assemble into virus-like
particles in insect cells (Belyaev and Roy, 1993). Exceptionally high
levels of expression is, perhaps, the most distinguishing feature of this
system. The highest level reported represents more than 50% of the
total cellular protein corresponding to approximately 1 gram of protein
product per 1 o9 cells (per 1 liter culture). Most heterologous proteins,
however, are produced at levels ranging from 10-100 mg per 1 o9 cells.
The use of very late promoters has a clear advantage of minimum
selective pressure on the virus to mutate towards heterologous gene
deletion or inactivation. Since baculoviruses are propagated at 270C,
the system can be used to produce large quantities of active protein
encoded by temperature sensitive alleles of a gene (Reynisdottir et a/.,
1990). The nucleocapsids of baculoviruses can accommodate 100 kbp of
additional DNA or more. Baculovirus vectors are helper-virus
independent and, therefore, relatively simple to use. All these reasons·
cited above help us to understand why BEVS has grown to become the
most powerful and popular expression system. The immense popularity
of BEVS is evident from the fact that more than 500 genes from
viruses, bacteria, fungi, plants, and animals have already been
expressed in this system (O'Reilly et a/., 1992).
However, the extent of glycosylation of some heterologous gene
products, such as tissue plasminogen activator, which require extensive
N-glycosylation, appears to decline late in infection (Jarvis and
Summers, 1989). This is probably due to an apparent decline in the
function of the endoplasmic reticulum due to a decline in host protein
synthesis during the late phase of infection. Therefore, an
overexpressed heterologous protein may not be post translationally
modified very well in this system due to the decline in functioning and
Introduction 4
because of the short time available prior to cell death caused by the
lytic nature of the virus. This .. secretory load.. is a' result of the
inability of the cells to cope with the processing of a large excess of
protein synthesized under the transcriptional control of a very late
promoter (Sridhar et a/., 1993a; Sridhar et a/., 1994). The use of a
promoter activated earlier in the infection cycle solves this problem of
.. secretory load .. to a large extent (Sridhar, 1993; Sridhar et a/., 1993b;
Hasnain et a/., 1994; Sridhar et a/., 1994).
The method of selection for recombinant viruses has evolved to a
great degree over the last few years. Traditionally, recombinants were
generated by in vivo recombination at the polh locus - two point
cross-overs between the viral DNA and a transfer vector carrying the
foreign gene flanked by viral sequences resulting in recombinant
progeny viruses having a replacement of the polh gene with the foreign
gene. These recombinants, however, appear at a very low frequency
(0.1-1 %) making microscopic screening (for polyhedrin negative
plaques) a very tedious process. Alternative methods to facilitate
screening include use of co-expressed reporter genes such as lacZ
(Weyer et a/., 1990) and luciferase (Hasnain et a/., 1994), antibody
screening, and PCR screening (O.Reilly et a/., 1992). Recently, a highly
efficient method which gives extraordinarily high rates of
recombination (>95%) has been reported (Kitts and Possee, 1993). Here,
an essential gene function adjacent to the polh locus, corresponding to
ORF 1629, is deleted from the wild type virus used for co-transfection.
This gene function can be complemented only after recombination with
a transfer vector that carries the foreign gene along with the deleted
complement of the viral gene thereby making it virtually impossible for
wild type progeny viruses to appear.
Introduction 5
2. Baculovirus Infection Process
AcNPV has a double-stranded, covalently closed, and circular DNA
genome of 128 kbp which has been completely sequenced (Ayres et a/.,
1994). The DNA is condensed into a nucleoprotein core which is present
within an enveloped capsid. These nucleocapsids are made in the
nucleus of infected cells. Two biochemically and morphologically
distinct progeny virus forms are produced during the infection process:
i) Budded viruses (BV), which are released into the extra cellular fluid
with a loosely fitting membrane envelope, and ii) Occluded virus (OV),
consisting of enveloped nucleocapsids embedded in a crystalline matrix
composed mainly of a protein called polyhedrin (Harrap, 1972). The
polyhedrin matrix protects the virus particles from inactivation by
physical factors after they are released into the environment.
Progress of infection in the insect: Insect larvae get infected
when they ingest OVs as contaminants of their food. The polyhedrin
matrix is solubilized in the midgut of the insects, releasing the virions
which enter midgut cells by fusion with the membrane of microvilli
(Granados and Williams, 1986). Infection of the polarized midgut cells
results in BV release from the basement membrane side of the cell
(Keddie et a/., 1989). This BV can gain access to the hemocoel and is
transported via the hemolymph to other tissues. BV released from the
midgut cells also infect epithelial cells of tracheoles, which provide
oxygen to the midgut, spreading infection along the tracheal network.
OVs produced by the infected tissues spread the infection to other
larvae after disintegration of the host.
Progress of Infection in Cell Culture: For repli.cation in cell
culture, the infection cycle occurs in three basic phases: early, late,
Introduction 6
and very late.
Early Phase - During this phase (approximately the first 6 hours
of infection) the cell is reprogrammed for virus replication. Infection in
cell culture is mediated by BVs entering by adsorptive endocytosis.
Nucleocapsids migrate through the cytoplasm into the nucleus where
the nucleoprotein core is released (Granados and Williams, 1986). There
is no requirement of viral protein synthesis for early gene transcription
(Nissen and Friesen, 1989). However, many early promoters are
transactivated by the product of the viral /E-1 gene (Guarino and
Summers, 1986) which is believed to enter the cell as a component of
the virions (Miller, 1988). Early gene transcription is thought to be
mediated by hos.t RNA polymerase II because no viral gene expression is
required and early gene transcription is sensitive to a-amanitin (Grula
et a/., 1981; Huh and Weaver, 1990).
Late phase - The late phase extends from 6 h pi (hours post
infection) to approximately 20 h pi. This is a period of extensive viral
DNA replication, late gene expression, and BV production. The A eN PV
genome has six . interspersed homologous regions, designated hr 1 to h r
5 (hr 4 contains two distinct hrs, designated hr 4a and hr 4b), of
approximately 500-800 bp in length that are believed to function as
origins of replication (Kool et a/., 1993).
Very late phase - This phase begins around 20 h pi.The polh
gene is· tiypertranscribed during this phase and the enveloped
nucleocapsids are embodied within the polyhedrin matrix of occlusion
bodies. Lysis of infected cells begins about 60 h pi, and by 72 h pi most
cells are in the process of dying and/or lysing.
Late and very late viral gene transcription, however,.· is
insensitive to a-amanitin (Grula et a/., 1981; Huh and Weaver, 1990).
Biochemical evidence suggests· that a new RNA polymerase is present in
Introduction 7
virus-infected cells which could be a virus-modified host RNA
polymerase, a new RNA polymerase with virus-encoded subunits, or
some combination of these two possibilities (Fuchs et a/., 1983; Yang e t
a/., 1991 ). Late and very late transcription is dependent on early viral
gene expression and on DNA replication -both cycloheximide and
aphidicolin block transcription of late and very late genes (Rice and
Miller, 1986).
Virus effects on host gene expression: AcNPV infection results in
shut-off of host gene expression, though host chromatin structure
appears to remain largely intact. A decline in steady-state levels of
host mRNAs begins approximately 12 h pi. By 24 h pi, steady-state
levels of cellular mRNAs such as actin and histone are quite low (Ooi
and Miller, 1988). Decline in host protein synthesis starts by 18 h pi,
and shut-off is virtually complete by 24 h pi (Carstens et a/., 1979). By
24 h pi, gene expression is primarily, if not exclusively, viral-specific.
The mechanism(s) by which host RNA and protein levels are down
regulated are not known. The virus encodes a protein (p35) that may be
responsible for shutting off cellular apoptotic gene function(s) thereby
enabling the virus to complete its own life cycle ending in lysis of the
host cells (Sugimoto et a/., 1994).
3. Genes Involved in Viral Transcription and Replication
Transcription regulatory genes: Baculovirus genes are transcribed
in a regulated cascade corresponding to the a, p, y, and o temporal
stages. The ·gene products of one temporal class activate, directly or
indirectly, the products of the next temporal class. Several genes that
appear to be involved in gene regulation have been identified. These
Introduction 8
genes include those encoding IE-0, IE-1, lE-N, PE-38, and CG30 (O'Reilly
et a/., 1992). IE-0 is actually an exon of one of the two forms of IE-1.
IE-1 is able to transactivate some early promoters in transient
expression assays (Guarino and Summers, 1986). The other three
proteins, lE-N, PE-38, and CG30, all share common unique structural
motifs: an unusual, double zinc-fingerlike motif and a C-terminal
leucine zipper. The unusual zinc-fingerlike motif is found in other
proteins, which are thought to have DNA-related functions (Free mont e t
a/., 1991 ). Thus, baculoviruses appear to have a family of related genes
that are probably involved in gene regulation.
Genes involved in DNA replication: Genes that are involved in viral
DNA replication include dnapo/, he/, and pcna. Enzymatic studies suggest
that not only is a new DNA polymerase activity induced during infection
but a host DNA polymerase activity is also stimulated (Miller et a/.,
1981 ). The dnapol gene (Tomalski et a/., 1988) encodes a 114-kDa
protein with homology to other DNA polymerases (eg., DNA polymerases
of herpes viruses, poxviruses, and adenoviruses). It is likely that this
gene encodes the AcNPV-induced, aphidicolin-sensitive DNA polymerase
described by Miller et a/. (1981 ). PCNA (proliferating-cell nuclear
antigen) may serve as a processivity factor for one of the DNA
polymerases. The he/ gene codes for a 143-kDa protein that contains a
region sharing sequence homology with other proteins having ATP
dependent helicase activity (Lu and Carstens, 1991 ).
4. Baculovirus Host Range and Choice of cells
Baculovirus Host Range: Baculoviruses have a narrow host range
with each baculovirus being able to infect only a few taxonomically
Introduction 9
related insect species (Groner, 1986). In a controlled survey done by
Bishop et a/. (1988) it was seen that A cNPV, which is considered to
have a relatively broad host range for a baculovirus, nevertheless, has a
highly restricted host range. A cNPV can infect about 40 different
Lepidopteran species belonging to 11 families and it can also
additionally infect a Coleopteran cell line (Croizier et a/., 1994). The
silkworm Bombyx mori is not the natural host for AcNPV. However,
Morris and Miller (1993) have reported .. non-productive., infections of
Bombyx mori cells with A cNPV i.e. there is no evident polyhedra
formation though there are low levels of late gene expression. They
conclude that .. non-productive.. infections in non-permissive cell lines
are abrogated in a number of different ways and no one common
restriction point preventing productive infection is evident. A 79
nucleotide region within the baculovirus p143 helicase gene has been
identified to play an important role in conferring species specificity
(Croizier et a/., 1994). A hybrid virus which can infect both Bombyx
mori and Spodoptera frugiperda cells has been constructed by allowing
homologous recombination between AcNPV and BmNPV (Mori et at.,
1992).
According to studies conducted by Carbonell et a/. (1985) the
restriction to AcNPV infection in many non-permissive insects is not
the inability to enter those host cells but its inability to replicate its
DNA and express late gene products. This is confirmed by a study of
factors blocking late gene transcription (Rice and Miller, 1986).
However, there is almost no literature available on experiments carried
out to delimit factors controlling the ability of different insect cell
lines to support AcNPV DNA replication and late gene transcription.
Choice of Cells: The cells most commonly used with A eN PV -based
vectors are Sf9 and Sf21 cells. Both these cell lines are derived from
Introduction 1 0
pupal ovarian tissue of Spodoptera frugiperda (the fall armyworm),
(Vaughn et a/., 1977). Sf9 is a clonal isolate of Sf21. Other cell lines
that support replication of AcNPV are available. TN368 is a cell line
derived from minced adult ovaries of Trichoplusia ni (the cabbage
lo,oper) and this cell line is also used for expression vector work (Hink,
1970). Two cell lines, BmN and Bm5, derived from Bombyx mori or the
common silk worm are commonly used for infection with the Bombyx
mori nuclear polyhedrosis virus (BmNPV), the other baculovirus which
is also used for expression work. BmN and Bm5 are derived from pupal
ovarian cells (O'Reilly et a/., 1992).
5. Regulation of the Polyhedrin Gene Promoter
The polh promoter has been the "workhorse" promoter of BEVS and. is
one of the strongest promoters to be used in any expression vector
system.
Unusual structure of the polyhedrin promoter: The po/h promoter
of AcNPV has been extensively characterized by deletion and linker
scan mutation analyses (Matsuura et a/., 1987; Possee and Howard,
1987; Ooi et a/., 1989). The transcription start point (Fig. 1) is at -50
relative to the A of the ATG translation start site (designated as +1 )
and lies within a highly conserved octanucleotide motif TAAGTATT.
This motif has been shown to be absolutely essential for transcription
initiation and the promoter has been defined as a 69 bp str~tch from -1
to -69. The sequence of the polh promoter is well conserved between
· otherwise distantly related baculoviruses (Rohrmann, 1986).
Mostly, bacterial and nuclear eukaryotic promoters have multiple
non-contiguous blocks of transcriptionally-important sequences
Introduction
-70 -50 +1
-----AATAAA I TAAGTATTI-1----------ATG
FIG. 1: Schematic representation of the AcNPV polyhedrln gene promoter. The essential polh promoter extends from -1 to -69 (the translation start point is designated as +1). The transcription start point. marked with a bent arrow, is at -50 and lies within a conserved, transcriptionally important TAAGTATT motif (boxed). The PPBP-cognate motifs (Burma et al., 1994) - AATAAA & TAAGTATT - have been shown. The 18 bp 'minimal' promoter (Morris and Miller, 1994) has been highlighted by a hatched bar.
Introduction 1 1
present upstream or downstream to the transcription start point. In
contrast, the polh promoter has an unusual structure and is similar only
to certain yeast mitochondrial and bacteriophage T7 and T3 promoters
where there is a conserved, transcriptionally important motif at the
transcription start point (Masters et a/., 1987}. Also, other
determinants of promoter activity are present in the untranslated
mRNA leader region (Ooi et a/., 1989) which is another unusual feature
of this promoter.
Recently, Morris and Miller (1994) have shown that. an 18 bp
sequence surrounding the transcription start point (Fig. 1) is sufficient
for minimal promoter activity, whereas the sequences encoding the
untranslated mRNA leader are required for the very-late burst of
expression. The structure of the polh promoter is, therefore, similar to
that of certain eukaryotic TAT A-less promoters where the 'initiator'
(the sequence around the transcription start site) acts as a 'minimal'
promoter capable of directing basal levels of transcription (Smale and
Baltimore, 1989; Weis and Reinberg, 1992).
The LEF genes and polyhedrin promoter activity: Seven early
genes, designated /ef-1, /ef-2, lef-3, lef-4, /ef-5, /ef-6, and /ef-7 (for
late expression factor) have been shown to be essential but not
sufficient for late gene expression. (Li et a/., 1993; Passarelli and
Miller, 1993a, b, c; Morris et a/., 1994; Passarelli and Miller, 1994}. The
level (replication, transcription, or translation) at which these genes
act is, however, not known. In fact, Kool et a/. (1994) have suggested
that /ef-1 and /ef-2 may be involved directly in DNA replication and,
therefore, indirectly in polh gene transcription.
hr1 enhances transcription from the polyhedrin promoter: The
AcNPV homologous region hr 1, located -3.7 kb upstream of the po/h
Introduction 12
promoter, has been shown to enhance transcription of a luciferase
reporter placed under the control of the polh promoter in transient
expression assays using AcNPV-infected Sf9 cells (Habib et a/., 1995).
This enhancer function of hr 1 has been shown to be independent of its
putative role as a origin of replication ( ori ) - Dpn I sensitivity assays
for DNA replication show that enhancement is not due to an increase in
plasmid copy number due to the ori function of hr 1. Habib et a/.,
therefore, suggested that the two functions of hr 1 (ori and enhancer)
may be carried out by different sequence elements within the hr 1
region.
A putative negative regulator binding immediately upstream
to the polyhedrin promoter: Etkin et a/. (1994) have reported the
identification of a 200-kDa host factor binding specifically to the -72
to -86 region of the polh promoter of AcNPV. This binding activity was
found in uninfected cells and also in cells during early stages of viral
infection, but decreased by 18 h p.i. Preliminary experiments suggested
that this protein functions as a negative regulator and the virus may
utilize this factor to control the differential expression of late and
very late genes.
PPBP - An unusual factor interacting with the polyhedrin
promoter: Gel retardation assays using nuclear extracts from A eN PV
infected Sf21 cell line revealed a 30-kDa host factor (polyhedrin
promoter-binding protein or PPBP) that binds to the po/h promoter
(Burma eta/., 1994}. A hexanucleotide sequence (AATAAA) within the
promoter is important for binding in association with the
transcriptionally essential TAAGTA TT motif (Fig. 1 ). PPBP has been
affinity purified to homogeneity and appears to be an unusual DNA
binding protein with respect to its stability (binds between 0.2 to 2 M
Introduction 1 3
NaCI and at temperatures as high as 650C), high binding affinity (binds
even in the absence of non-specific DNA with an apparent dissociation
constant of -3.7 x 1 o-12 M) and high specificity of binding (binds in the
presence of a 50,000-fold excess of non-specific DNA). Phosphorylation
of this factor is essential for its DNA-binding activity. Interestingly,
the PPBP-cognate region corresponds to the minimal promoter
described by Morris and Miller (1994), (Fig. 1 ). The correlation between
functional promoter sequences and the factor-binding site argues that
the interaction of PPBP with the polh promoter must be an important
event in the activation of transcription from this promoter. It has been
proposed that PPBP might function in a manner analogous to the TATA
binding protein in recruiting the virus-specific RNA polymerase and/or
basal transcription factor(s) to the polh promoter.
6. Aims and Objectives
The level of synthesis of heterologous proteins in BEVS varies
considerably and is thought to be due to a number of factors (O'Reilly e t
a/., 1992; Hasnain et a/., 1994; Sridhar et a/., 1994; Ranjan and Hasnain,
1995). Efforts to increase the level of expression of foreign genes in
baculovirus systems have been directed at optimizing the locations of
foreign genes in relation to transcriptional and translational signals
within the polh gene. There has been very little attention directed at
the influence of the host cell line on protein expression. Significant
levels of cell line-dependent differences in reporter gene expression
have been observed in a study carried out by Hink et a/. (1991 ). However,
the molecular mechanism(s) governing such cell line-dependent
differences in expression have not been worked out. The number of
insect cells which support productive AcNPV infections is very limited
Introduction 14
(Morris and Miller, 1992). Characterization of the nature of the block(s)
to productive AcNPV infection in refractive insect cells may provide
insights into baculovirus-host cell interactions and host range
determination. Understanding the molecular mechanism(s) that
determine the selectivity of baculovirus replication at the cellular
level would facilitate the assessment of recombinant baculovirus
safety to non-target organisms and might allow control of the host
range of these viruses. In order to address these questions we used a
recombinant baculovirus vAcBhCG-Iuc that carries the genes for BhCG
and firefly luciferase cloned under two independent copies of the polh
promoter (Jha et a/., 1992; Hasnain et a/., 1994). The expression of
these two reporter proteins was studied in a panel of insect cell lines
and studies were carried out to determine the molecular mechanism(s)
governing differential expression and blocks to productive infections.
As a first step, evidences were collected to determine the level at
which such cell line-dependent expression of foreign genes may be
affected.
I nspite of the fact that this system is widely used for
heterologous expression, very little is known about the regulation of
the polh promoter and the mechanism responsible for hyper
transcription from this promoter. The only factor (PPBP) directly
interacting with the polh promoter has been recently identified and
characterized (Burma et a/., 1994). Given the importance of this unusual
factor,. the interaction of PPBP with the polh promoter in permissive
as well as non-permissive cell lines and with respect to differential
expression of foreign genes has not been worked out. We, therefore,
carried out experiments to study the interaction of PPBP with the polh
promoter in permissive and non-permissive cell lines and the
differences, if any, in terms of such interactions between the over and
Introduction 1 5
under-expressed cell lines.
The unusual structure of the polh promoter (with the 'initiator'
acting as the 'minimal' promoter) and the binding of PPBP at the
transcription start point poses the problem of whether binding would be
maintained even after melting of DNA at this point during transcription
initiation. We, therefore, carried out experiments to · investigate
whether PPBP exhibits any single-stranded DNA-binding activity. Such
an activity would allow PPBP to maintain its position when the DNA
helix melts during th~ initiation of transcription.
The objectives of the study described in this thesis, therefore, can be
summarized as follows:
1 ) To study levels of f3hCG and luciferase expression in a panel of
insect cell lines infected with a dual recombinant virus vAcBhCG
/uc.
2) To study molecular mechanism(s) governing cell line ... dependent
differences in expression with respect to:
a) Virus entry
b) Virus replication
c) Transcription of reporter genes
d) Reporter mANA stability
3) To study DNA-protein interactions at the polh promoter in
permissive and non-permissive cell lines.
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