virus genus - virology educationregist2.virology-education.com/1xmrv/docs/01_coffin.pdf ·...

Presented at the 1st Intl. Workshop on XMRV7-8 September, Bethesda USA

Retroviruses:Endogenous MLV and XMRV

John M. CoffinTufts University

Retroviruses:Endogenous MLV and XMRV

John M. CoffinTufts University

Tufts University

dut

orfA, orfB, orfC

new env

bel1, bel2

Virus Genus

MLVFeLVHERV-CWDS

tat, rev

sag

tax, rex

HFV

HIV-1HIV-2EIAVVMV

MPMV

MMTVHERV-KIAP

ASLV

BLVHTLV-1HTLV-2

Deltaretrovirus

The Retrovirus Family Tree

Epsilonretrovirus

Gammaretrovirus

Betaretrovirus

Alpharetrovirus

Gammaretroviru

Betaretrovirus

Alpharetrovirus

New GenesAt least 30 million years ago!

LentivirusLentivirus

Spumaretrovirinae



Endogenous RetrovirusesEndogenous Retroviruses

1. Remnants of germ line infections by exogenous (infectious) retroviruses.1. Remnants of germ line infections by exogenous (infectious) retroviruses.

2. Became fixed in the host species.Some confer protection against future infections by the same or similar viruses. A few others have salutary effects.

2. Became fixed in the host species.Some confer protection against future infections by the same or similar viruses. A few others have salutary effects.

5. Comprise 6-8% of the human genome. (More viruses than us).5. Comprise 6-8% of the human genome. (More viruses than us).

3. Inherited like normal genes.3. Inherited like normal genes.

4. Present in every vertebrate and many invertebrates.4. Present in every vertebrate and many invertebrates.


Endogenous RetrovirusesEndogenous Retroviruses

6. Provide a fossil record of pathogen-host interaction unavailable in any other system.

7. Can participate in evolutionary processes as well as inform us about them.

8. Involved in disease in some animals. Humans?

6. Provide a fossil record of pathogen-host interaction unavailable in any other system.

7. Can participate in evolutionary processes as well as inform us about them.

8. Involved in disease in some animals. Humans?


XMRVXMRV

1. First described about 5 years ago in a few patients with prostate cancer.2. Within the last year:

• Reported in 25% of prostate cancer biopsies.• Reported in 67% of chronic fatigue syndrome patients.• Reported in 4% of normal controls in both studies.• Not always found in studies from other labs on samples from either

disease.

3. Isolates from the 2 diseases at different times and locations are almost identical to one another.

4. Causality, prevalence, and distribution remain to be established.5. Very closely related to endogenous xenotropic (X) MLV.6. Recently, Lo et al (PNAS) reported also finding MLV-like (PMV and

MPMV related) virus sequences associated with CFS.

1. First described about 5 years ago in a few patients with prostate cancer.2. Within the last year:

• Reported in 25% of prostate cancer biopsies.• Reported in 67% of chronic fatigue syndrome patients.• Reported in 4% of normal controls in both studies.• Not always found in studies from other labs on samples from either

disease.

3. Isolates from the 2 diseases at different times and locations are almost identical to one another.

4. Causality, prevalence, and distribution remain to be established.5. Very closely related to endogenous xenotropic (X) MLV.6. Recently, Lo et al (PNAS) reported also finding MLV-like (PMV and

MPMV related) virus sequences associated with CFS.


GammaretrovirusesGammaretroviruses1. Simple retroviruses (only gag, pol, and env genes).

2. Widespread in nature, with isolates from mammals (mice, cats, primates, koalas and others), birds, and reptiles.

3. Readily give rise to endogenous proviruses.

4. Most commonly transmitted vertically (mother to offspring).

5. Cause a wide variety of diseases (cancer, neurological, degenerative, immunodeficiency).

6. Cancer is usually the result of insertional inactivation of protooncogenes.

7. Infection is lifelong, with persistence of infected cells established early, and-unlike HIV, very few replication cycles per host.

1. Simple retroviruses (only gag, pol, and env genes).

2. Widespread in nature, with isolates from mammals (mice, cats, primates, koalas and others), birds, and reptiles.

3. Readily give rise to endogenous proviruses.

4. Most commonly transmitted vertically (mother to offspring).

5. Cause a wide variety of diseases (cancer, neurological, degenerative, immunodeficiency).

6. Cancer is usually the result of insertional inactivation of protooncogenes.

7. Infection is lifelong, with persistence of infected cells established early, and-unlike HIV, very few replication cycles per host.


Xenotropic MLVXenotropic MLV1. Inherited as endogenous proviruses (ca 10-20 copies) in all inbred mice.

2. Many more proviruses in some wild Mus Musculus subspecies.

3. Some proviruses (e.g., Bxv1-XMV43) are intact and infectious.

4. Can infect virtually all mammals, except some species of Mus, due to receptor (Xpr1) polymorphism.

5. Not directly pathogenic in mice (due to lack of receptor), but LTR is often found in oncogenic recombinant MLVs.

6. Pathogenicity in other species is unknown.

7. Common contaminant of human tumor cell lines due to passage through nude mice (which have Bxv1).

8. Closely related to XMRV, but none is identical, perhaps excluding inbred mice as the origin.

9. Related MLVs cause a wide variety of diseases (malignant, immunodeficiency, neurological) in mice.

1. Inherited as endogenous proviruses (ca 10-20 copies) in all inbred mice.

2. Many more proviruses in some wild Mus Musculus subspecies.

3. Some proviruses (e.g., Bxv1-XMV43) are intact and infectious.

4. Can infect virtually all mammals, except some species of Mus, due to receptor (Xpr1) polymorphism.

5. Not directly pathogenic in mice (due to lack of receptor), but LTR is often found in oncogenic recombinant MLVs.


7. Common contaminant of human tumor cell lines due to passage through nude mice (which have Bxv1).

8. Closely related to XMRV, but none is identical, perhaps excluding inbred mice as the origin.

9. Related MLVs cause a wide variety of diseases (malignant, immunodeficiency, neurological) in mice.


Polytropic and Modified Polytropic MLVPolytropic and Modified Polytropic MLV1. Inherited as endogenous proviruses (ca 10-20 copies each) in all inbred

mice.

2. Also found in wild Mus Musculus subspecies.

3. No infectious virus has been found to date.

4. Can infect virtually all mammals, using both forms of the Xpr1 Receptor.

5. Not directly pathogenic in mice (due to lack of infectivity?), but env gene is often found in oncogenic recombinant MLVs.


7. Both types derived independently from XMV, probably in response to Xpr1 mutation.

8. Unlike XMV, PMV and MPMV proviruses have suffered significant mutations mediated by mouse APOBEC3.

1. Inherited as endogenous proviruses (ca 10-20 copies each) in all inbred mice.

2. Also found in wild Mus Musculus subspecies.

3. No infectious virus has been found to date.

4. Can infect virtually all mammals, using both forms of the Xpr1 Receptor.

5. Not directly pathogenic in mice (due to lack of infectivity?), but env gene is often found in oncogenic recombinant MLVs.


7. Both types derived independently from XMV, probably in response to Xpr1 mutation.

8. Unlike XMV, PMV and MPMV proviruses have suffered significant mutations mediated by mouse APOBEC3.

Formation of Endogenous Proviruses

provirus

Long terminal repeats(LTR’s)Identical sequence

Endogenous provirusPresented at the 1st Intl. Workshop on XMRV7-8 September, Bethesda USA

Host-Retrovirus Evolution


Effects of Endogenous Proviruses(Good and Bad)

Syncytinenv


B H B B

B B B E

B B B E

B B H B E

pEco

js6/10

js5

js4

Ecotropic

Xenotropic

Polytropic

Modifiedpolytropic

B

Selectivehybridization probe Insert in LTR

Four Classes of Endogenous MLV


Specific restriction sites

Specific hybridization probe

Detection of Specific Proviruses

ProbeA BA B

Size of fragments detected depends on location of cleavage site in flanking DNA, one band per provirus.


MpmvPmv Xmv

B C Ak D H A L B H Ak D A L C B C Ak D H A L

Distribution of Endogenous Proviruses in Inbred Mice


Distribution of Endogenous MLV’s on Mouse Chromosomes

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 X Y

=Mtv=Pmv=Pltr

=Mpmv=Mltr =Xltr

=Xmv=Emv

=centromere=10cM



Oncogenesis by Endogenous MLVOncogenesis by Endogenous MLV

1. In AKR and some other inbred strains, most mice die within a year of thymic lymphoma.

2. The proximal cause is integration adjacent to one or more protooncogenes (c-myc, pim-1, and others) of a provirus derived from endogenous MLV.

3. Around the time of birth all mice express a replicating ecotropic virus (AKV).

4. A complex, but highly reproducible series of events leads to the formation of a recombinant virus containing a polytropic env gene and an LTR derived from bxv1 and modified by enhancer duplication.

1. In AKR and some other inbred strains, most mice die within a year of thymic lymphoma.

2. The proximal cause is integration adjacent to one or more protooncogenes (c-myc, pim-1, and others) of a provirus derived from endogenous MLV.

3. Around the time of birth all mice express a replicating ecotropic virus (AKV).

4. A complex, but highly reproducible series of events leads to the formation of a recombinant virus containing a polytropic env gene and an LTR derived from bxv1 and modified by enhancer duplication.

gag pol env

Akv

Bxv1

Pmv

gag env

Recombinant 1

Recombinant 2

MCF

Viruses generated during leukemogenesis

gag env

gag env

pro

polpro

polpro

polpro

gag envpolpro

gag envpolpro

Generation and selection of Recombinant Proviruses in AKR Leukemia


a b c d e f g h i j k a b c d e f g h i j ka b c d e f g h i j kA B C

X-I (KT-55) X-II (KT-51) X-III (KT-53)

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Endogenous MLVs in Lab and Wild Mice


M. Spicelegus (formerly M. hortulanus)

Lab strains

HEM

X-I

ecotropic

P-I

P-IIP-III

P-IV

P-V

X-II

X-IV

1-24-6 3-5

MYA

X-III

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0

Coevolution of Endogenous MLV’s and Mice

M. Spretus

M. m. molissinus

M. m. domesticus

M. m. musculus

M. m. castaneus



Distribution of Endogenous Proviruses in Inbred Mice

Mpmv Xmv

C57BL/6J

Pmv

Relationship of Proviruses in the Sequenced Genome

XMRV (Lombardi et al.,Science 2009)

MLV-like(Lo et al.,PNASTwo weeks ago)



Endogenization

Receptor Mutation

CFS pt 1010CFS pt 1042PC VP 62PC VP 42PC VP 35

XMV13 ProvirusNZB Xenotropic MLV

AKR MLV

2%

BALB/c Xenotropic MLV

Moloney MLV

Exogenous MLV

Relationship of XMRV to Endogenous MLVs

Xenotropic MLV

Endogenous MLV

Virus Mutation

Polytropic MLV

XMRV

MLV-like


XMRV vs MLV-LikeXMRV vs MLV-Like

1. XMRV (Urisman et al, Lombardi et al, others):1. Isolated as infectious virus from Prostate Cancer, CFS patients.2. Isolates are very similar in sequence.3. They form a distinct subclade among XMV proviruses.

2. MLV-like sequences (Lo et al)1. PCR amplified using MLV gag and env primers.2. Found preferentially in CFS plasma.3. No infectious virus yet identified.4. Only fragmentary, bulk, sequences available at present.

3. Until the relationship between the two is further clarified, it is important to consider these to be distinct, unrelated phenomena.

1. XMRV (Urisman et al, Lombardi et al, others):1. Isolated as infectious virus from Prostate Cancer, CFS patients.2. Isolates are very similar in sequence.3. They form a distinct subclade among XMV proviruses.

2. MLV-like sequences (Lo et al)1. PCR amplified using MLV gag and env primers.2. Found preferentially in CFS plasma.3. No infectious virus yet identified.4. Only fragmentary, bulk, sequences available at present.

3. Until the relationship between the two is further clarified, it is important to consider these to be distinct, unrelated phenomena.


Why I Don’t Sleep Well at NightWhy I Don’t Sleep Well at Night

1. We can be optimistic that XMRV will turn out to be important in the etiology of CFS and prostate cancer, but the case is not yet proven:

1. Conflicting reports on the association of either disease with the virus2. Lack of insight into pathogenic mechanisms

2. Studies can be complicated by minute traces of mouse DNA or by viruses derived from lab strain mice.

3. One mouse cell contains over 100 PMV and XMV proviruses. Sensitive PCR assays can detect the provirus in 10 fg of mouse DNA.

4. It’s well known that xenotropic MLVs derived from nude mice have inadvertently spread through many labs.

5. While the major XMRV studies published to date appear to be wellcontrolled for these problems, extraordinary caution is necessary.

1. We can be optimistic that XMRV will turn out to be important in the etiology of CFS and prostate cancer, but the case is not yet proven:

1. Conflicting reports on the association of either disease with the virus2. Lack of insight into pathogenic mechanisms

2. Studies can be complicated by minute traces of mouse DNA or by viruses derived from lab strain mice.

3. One mouse cell contains over 100 PMV and XMV proviruses. Sensitive PCR assays can detect the provirus in 10 fg of mouse DNA.

4. It’s well known that xenotropic MLVs derived from nude mice have inadvertently spread through many labs.

5. While the major XMRV studies published to date appear to be wellcontrolled for these problems, extraordinary caution is necessary.


Why I Don’t Sleep Well at NightWhy I Don’t Sleep Well at Night

6. In subsequent talks, my colleagues from Tufts and the NCI will present our efforts to deal with this problem:

1. Mouse origin of XMRV and a sensitive assay for contamination (Cingöz)

2. Environmental contamination with mouse DNA (Huber)3. Sensitive assays for XMRV and endogenous MLVs (Kearney)

6. In subsequent talks, my colleagues from Tufts and the NCI will present our efforts to deal with this problem:

1. Mouse origin of XMRV and a sensitive assay for contamination (Cingöz)

2. Environmental contamination with mouse DNA (Huber)3. Sensitive assays for XMRV and endogenous MLVs (Kearney)


QuickTime™ and a decompressor

are needed to see this picture.



XMRVXMRVMice are Everywhere!Mice are Everywhere!





One drop of mouse blood in my swimming pool…

Equals One MLV provirus per ml.


XMRV, CFS, and AntiviralsXMRV, CFS, and Antivirals

1. Like other MLVs, XMRV is sensitive to inhibition by a few antiretrovirals licensed to treat HIV infection.

2. A survey of the blogs indicates that there may be significant off-label use of some of these compounds in CFS patients, with anecdotal results posted.

3. While there could be value in well-controlled randomized trials of these agents, we should all oppose uncontrolled dosing of patients with antivirals:

1. We do not know for sure that the virus is causal.2. The drugs can only work by blocking replication cycles, not reparing

damage already coused by virus replication.3. The disease lacks objective biomarkers to monitor treatment.4. There is no good assay to monitor effects on virus replication.5. Results of such studies will never be accepted by third-party payers or

regulators, keeping the treatment out of reach of the large majority of thosse suffering from CFS.

1. Like other MLVs, XMRV is sensitive to inhibition by a few antiretrovirals licensed to treat HIV infection.

2. A survey of the blogs indicates that there may be significant off-label use of some of these compounds in CFS patients, with anecdotal results posted.

3. While there could be value in well-controlled randomized trials of these agents, we should all oppose uncontrolled dosing of patients with antivirals:

1. We do not know for sure that the virus is causal.2. The drugs can only work by blocking replication cycles, not reparing

damage already coused by virus replication.3. The disease lacks objective biomarkers to monitor treatment.4. There is no good assay to monitor effects on virus replication.5. Results of such studies will never be accepted by third-party payers or

regulators, keeping the treatment out of reach of the large majority of thosse suffering from CFS.



AcknowledgementsAcknowledgements

Tufts University

- Jonathan Stoye-Wayne Frankel-Patric Jern-Oya Cingöz-Brigitte Huber

- Jonathan Stoye-Wayne Frankel-Patric Jern-Oya Cingöz-Brigitte Huber




AcknowledgementsAcknowledgements

-Mary Kearney-Ann Wiegand-Jon Spindler-Wei Shao

National Cancer Institute at FrederickNational Cancer Institute at FrederickHIV Drug Resistance ProgramHIV Drug Resistance ProgramHIV Drug Resistance Program


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