Retroviruses: Endogenous MLV and XMRV

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Ashlyn Eaton
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1 Retroviruses: Endogenous MLV and XMRV John M. Coffin Tufts University Tufts University

2 The Retrovirus Family Tree Virus Genus bel1, bel2 tat, rev orfa, orfb, orfc HFV MLV FeLV HERV-C WDS HIV-1 HIV-2 EIAV VMV Spumaretrovirinae Gammaretrovirus Epsilonretrovirus Lentivirus dut MPMV sag new env MMTV HERV-K IAP Betaretrovirus ASLV Alpharetrovirus tax, rex BLV HTLV-1 HTLV-2 Deltaretrovirus New Genes At least 30 million years ago!

3 Endogenous 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. 3. Inherited like normal genes. 4. Present in every vertebrate and many invertebrates. 5. Comprise 6-8% of the human genome. (More viruses than us).

4 Endogenous 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?

5 XMRV 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.

6 Gammaretroviruses 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, andunlike HIV, very few replication cycles per host.

7 Xenotropic MLV 1. Inherited as endogenous proviruses (ca 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.

8 Polytropic and Modified Polytropic MLV 1. Inherited as endogenous proviruses (ca 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. 6. Pathogenicity in other species is unknown. 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.

9 Formation of Endogenous Proviruses provirus Long terminal repeats (LTR s) Identical sequence Endogenous provirus

10 Host-Retrovirus Evolution

11 Effects of Endogenous Proviruses (Good and Bad) Syncytin env

12 Four Classes of Endogenous MLV Ecotropic B H B B B Xenotropic Polytropic peco B B B E BB B E js6/10 Modified polytropic BB H B E js5 js4 Selective hybridization probe Insert in LTR

13 Detection of Specific Proviruses Specific restriction sites Probe A B Specific hybridization probe Size of fragments detected depends on location of cleavage site in flanking DNA, one band per provirus.

14 Distribution of Endogenous Proviruses in Inbred Mice B C Ak D H A L Pmv Presented at the 1st Intl. Workshop on XMRV B H Ak D A L C Mpmv B C Ak D H A L Xmv

15 Distribution of Endogenous MLV s on Mouse Chromosomes =10cM =centromere =Emv =Pmv =Mpmv =Xmv =Mtv =Pltr =Mltr =Xltr X Y

16 Oncogenesis 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.

17 Generation and selection of Recombinant Proviruses in AKR Leukemia Akv gag pro pol env Bxv1 gag pro pol env Pmv gag pro pol env Viruses generated during leukemogenesis Recombinant 1 gag pro pol env Recombinant 2 gag pro pol env MCF gag pro pol env

Endogenous MLVs in Lab and Wild Mice A B C lab.

jk Bxv-1 Xmv28 Xmv10 X-I (KT-55) X-II (KT-51)

mol dom dom mus Mus. spr spr. spi spi. car cer.

lab cas mus mol dom lab cas mus mol dom dom dom

18 Endogenous MLVs in Lab and Wild Mice A B C lab. cas. mus. mol. dom. lab. cas. mus. mol. dom. lab. cas. mus. mol. dom. a bcdef gh i j k Kb a bcdef gh i j k a bcdef ghi jk Bxv-1 Xmv28 Xmv10 X-I (KT-55) X-II (KT-51) X-III (KT-53) mus. spr. hor. cer. car. coo. lab. cas. mus. mol. dom. lab lab cas. cas mus. mus mol. dom dom D a bcdef ghi jk lmnopqr s t E lab. cas. mus. mol. dom. lab lab cas. cas mus. mus mol. mol dom dom mus Mus. spr spr. spi spi. car cer. coo car. cer coo. lab cas mus mol dom lab cas mus mol dom lab lab cas. cas mus. mus mol. dom dom abcdefghi jk X-IV (KT-59/60) Xltr

19 Coevolution of Endogenous MLV s and Mice HEM M. Spicelegus (formerly M. hortulanus) M. Spretus X-IV P-V P-I P-IV P-II P-III M. m. domesticus } M. m. musculus M. m. molissinus M. m. castaneus Lab strains X-I X-II X-III ecotropic MYA

20 Distribution of Endogenous Proviruses in C57BL/6J Inbred Mice Pmv Mpmv Xmv

21 Relationship of Proviruses in the Sequenced Genome MLV-like (Lo et al., PNAS Two weeks ago) XMRV (Lombardi et al., Science 2009)

22 Relationship of XMRV to Endogenous MLVs Exogenous MLV Endogenization Endogenous MLV Receptor Mutation Xenotropic MLV CFS pt 1010 CFS pt 1042 PC VP 62 PC VP 42 PC VP 35 XMV13 Provirus NZB Xenotropic MLV BALB/c Xenotropic MLV AKR MLV Moloney MLV 2% Virus Mutation XMRV Polytropic MLV MLV-like

23 XMRV 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.

24 Why 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 virus 2. 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 well controlled for these problems, extraordinary caution is necessary.

25 Why 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)

26 Mice are Everywhere! XMRV QuickTime and a decompressor are needed to see this picture. QuickTime and a decompressor are needed to see this picture.

27 One drop of mouse blood in my swimming pool Equals One MLV provirus per ml. QuickTime and a decompressor are needed to see this picture.

28 XMRV, 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.

29 Acknowledgements - Jonathan Stoye -Wayne Frankel -Patric Jern -Oya Cingöz -Brigitte Huber QuickTime and a decompressor are needed to see this picture. Tufts University

30 Acknowledgements -Mary Kearney -Ann Wiegand -Jon Spindler -Wei Shao QuickTime and a decompressor are needed to see this picture. HIV Drug Resistance Program National Cancer Institute at Frederick

Retroviruses. ---The name retrovirus comes from the enzyme, reverse transcriptase.

Retroviruses. ---The name retrovirus comes from the enzyme, reverse transcriptase. Retroviruses ---The name retrovirus comes from the enzyme, reverse transcriptase. ---Reverse transcriptase (RT) converts the RNA genome present in the virus particle into DNA. ---RT discovered in 1970.