The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Infectivity assessment of porcine endogenous retrovirus using high-throughput sequencing technologies.

Xenogenic cell-based therapeutic products are expected to alleviate the chronic shortage of human donor organs. For example, porcine islet cell products are currently under development for the treatment of human diabetes. As porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the case of products that use living pig cells as raw materials. Although several PERV sequences exist in the porcine genome, not all have the ability to infect human cells. Therefore, polymerase chain reaction analysis, which amplifies a portion of the target gene, may not accurately assess the infection risk. Here, we determined porcine genome sequences and evaluated the infectivity of PERVs using high-throughput sequencing technologies. RNA sequencing was performed on both PERV-infected human cells and porcine cells, and reads mapped to PERV sequences were examined. The normalized number of the reads mapped to PERV regions was able to predict the infectivity of PERVs, indicating that it would be useful for evaluation of the PERV infection risk prior to transplantation of porcine products.

Koala retrovirus diversity, transmissibility, and disease associations.

BACKGROUND: Koalas are infected with the koala retrovirus (KoRV) that exists as exogenous or endogenous viruses. KoRV is genetically diverse with co-infection with up to ten envelope subtypes (A-J) possible; KoRV-A is the prototype endogenous form. KoRV-B, first found in a small number of koalas with an increased leukemia prevalence at one US zoo, has been associated with other cancers and increased chlamydial disease. To better understand the molecular epidemiology of KoRV variants and the effect of increased viral loads (VLs) on transmissibility and pathogenicity we developed subtype-specific quantitative PCR (qPCR) assays and tested blood and tissue samples from koalas at US zoos (n = 78), two Australian zoos (n = 27) and wild-caught (n = 21) in Australia. We analyzed PCR results with available clinical, demographic, and pedigree data. RESULTS: All koalas were KoRV-A-infected. A small number of koalas (10.3%) at one US zoo were also infected with non-A subtypes, while a higher non-A subtype prevalence (59.3%) was found in koalas at Australian zoos. Wild koalas from one location were only infected with KoRV-A. We observed a significant association of infection and plasma VLs of non-A subtypes in koalas that died of leukemia/lymphoma and other neoplasias and report cancer diagnoses in KoRV-A-positive animals. Infection and VLs of non-A subtypes was not associated with age or sex. Transmission of non-A subtypes occurred from dam-to-offspring and likely following adult-to-adult contact, but associations with contact type were not evaluated. Brief antiretroviral treatment of one leukemic koala infected with high plasma levels of KoRV-A, -B, and -F did not affect VL or disease progression. CONCLUSIONS: Our results show a significant association of non-A KoRV infection and plasma VLs with leukemia and other cancers. Although we confirm dam-to-offspring transmission of these variants, we also show other routes are possible. Our validated qPCR assays will be useful to further understand KoRV epidemiology and its zoonotic transmission potential for humans exposed to koalas because KoRV can infect human cells.

Host factors for retroviral integration site selection.

To achieve productive infection, retroviruses such as HIV stably integrate their reverse transcribed RNA genome into a host chromosome. Each retroviral family preferentially integrates near a unique subset of genomic features. HIV integrase (IN) is targeted to the body of active transcription units through interaction with lens epithelium-derived growth factor (LEDGF/p75). We describe the successful effort to develop inhibitors of the interaction between IN and LEDGF/p75, referred to as LEDGINs. Gammaretroviruses display a distinct integration pattern. Recently, BET (bromo- and extraterminal domain) proteins were identified as the LEDGF/p75 counterparts that target the integration of gammaretroviruses. The identification of the chromatin-readers LEDGF/p75 and BET as cellular cofactors that orchestrate lentiviral or gammaretroviral integration opens new avenues to developing safer viral vectors for gene therapy.

Coinfection with koala retrovirus subtypes A and B and its impact on captive koalas in Japanese zoos.

Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.

RD-114 virus story: from RNA rumor virus to a useful viral tool for elucidating the world cats' journey.

RD-114 virus is a feline endogenous retrovirus (ERV) isolated from human rhabdomyosarcoma in 1971 and classified as endogenous gammaretrovirus in domestic cats (Felis catus). Based on the previous reports in 70's, it has been considered that a horizontal, infectious event occurred to transfer the virus from ancient baboon species to ancient cat species, whereupon it became endogenous in the cat species about several million years ago in Mediterranean Basin. Although it has been believed that all domestic cats harbor infectious RD-114 provirus in their genome, we revealed that cats do not have infectious RD-114 viral loci, but infectious RD-114 virus is resurrected by recombination between uninfectious RD-114 virus-related ERVs [here we designated them as RD-114-related sequences (RDRSs)]. Further, we also revealed the RDRSs which would potentially be resurrected as RD-114 virus (here we refer to them as ''new'' RDRSs) had entered the genome of the domestic cat after domestication of the cat around 10 thousand years ago. The fractions and positions of RDRSs in the cat genome differed in Western and Eastern cat populations and cat breeds. Our study revealed that RDRS would be a useful tool for elucidating the world travel routes of domestic cats after domestication.

Human and murine APOBEC3s restrict replication of koala retrovirus by different mechanisms.

BACKGROUND: Koala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag. RESULTS: In this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results. CONCLUSIONS: These results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.

Early events in retrovirus XMRV infection of the wild-derived mouse Mus pahari.

A novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. Standard Mus musculus laboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whether Mus pahari could serve as a model for XMRV, primary Mus pahari fibroblasts and mice were infected with cell-free XMRV. Infection of cells in vitro resulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV into Mus pahari mice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP(+) CD4(+) T cells and CD19(+) B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3 in vivo. These data demonstrate infection of Mus pahari by XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infection in vivo. These data support the use of Mus pahari as a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.

Common inbred strains of the laboratory mouse that are susceptible to infection by mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV.

Laboratory mouse strains carry endogenous copies of the xenotropic mouse leukemia viruses (X-MLVs), named for their inability to infect cells of the laboratory mouse. This resistance to exogenous infection is due to a nonpermissive variant of the XPR1 gammaretrovirus receptor, a resistance that also limits in vivo expression of germ line X-MLV proviruses capable of producing infectious virus. Because laboratory mice vary widely in their proviral contents and in their virus expression patterns, we screened inbred strains for sequence and functional variants of the XPR1 receptor. We also typed inbred strains and wild mouse species for an endogenous provirus, Bxv1, that is capable of producing infectious X-MLV and that also contributes to the generation of pathogenic recombinant MLVs. We identified the active Bxv1 provirus in many common inbred strains and in some Japanese Mus molossinus mice but in none of the other wild mouse species that carry X-MLVs. Our screening for Xpr1 variants identified the permissive Xpr1(sxv) allele in 7 strains of laboratory mice, including a Bxv1-positive strain, F/St, which is characterized by lifelong X-MLV viremia. Cells from three strains carrying Xpr1(sxv), namely, SWR, SJL, and SIM.R, were shown to be infectable by X-MLV and XMRV; these strains carry different alleles at Fv1 and vary in their sensitivities to specific X/P-MLV isolates and XMRV. Several strains with Xpr1(sxv) lack the active Bxv1 provirus or other endogenous X-MLVs and may provide a useful model system to evaluate the in vivo spread of these gammaretroviruses and their disease potential in their natural host.

Xenotropic murine leukemia virus-related virus establishes an efficient spreading infection and exhibits enhanced transcriptional activity in prostate carcinoma cells.

Xenotropic murine leukemia virus-related virus (XMRV) is a novel human gammaretrovirus discovered in association with human prostate tumors. XMRV was first identified in prostate stromal cells surrounding the tumors of patients carrying a mutation in the HPC1 gene locus. To determine the tropism of XMRV in cell culture, we tested the ability of XMRV to spread and replicate in various prostate and nonprostate cell lines. We found that although the expression of XMRV viral proteins and the spread of infectious virus were minimal in a variety of cell lines, XMRV displayed robust expression and infection in LNCaP prostate tumor cells. The transcriptional activity of the XMRV long terminal repeat (LTR) was found to be higher than the Moloney murine leukemia virus LTRs in both LNCaP and WPMY-1 (simian virus 40-transformed prostate stromal cells). The U3 promoter of XMRV and a glucocorticoid response element (GRE) within the U3 were required for the transcriptional activity in LNCaP cells. Coexpression of the androgen receptor and stimulation with dihydrotestosterone stimulated XMRV-LTR-dependent transcription in 293T cells, and the GRE was required for this activity. These data suggest that XMRV may replicate more efficiently in LNCaP cells in part due to the transcriptional environment in LNCaP cells.

Effect of pseudotype on Abelson virus and Kirsten sarcoma virus-induced leukemia.

Nonproducer cells transformed by Kirsten sarcoma virus (KiSV) or Abelson murine leukemia virus (A-MuLV) were infected with N- or NB-tropic helper viruses to rescue the defective transforming virus. The titer of the transforming viruses was determined on NIH/3T3 fibroblast-like cells and cell-free filtrates of virus stock were inoculated into newborn Fv-1nn mice. Friend, Moloney, and Rauscher group of MuLV (FMR) pseudotypes of KiSV induced an erythroid leukemia efficiently, while an endogenous helper (N35-MuLV) pseudotype of KiSV did not. FMR pseudotypes of A-MuLV induced the Abelson lymphoid leukemia, while the N35-MuLV or a Kirsten leukemia virus (Ki-MuLV) pseudotype did not. Pseudotypes of A-MuLV were used to infect bone marrow cells of Fv-1nn mice in vitro. The FMR pseudotypes transformed bone marrow cells at 40-100-fold higher frequency than the N35-MuLV or Ki-MuLV pseudotypes. Mixing experiments demonstrated that the addition of an effective helper, such as M-MuLV did not enhance lymphoid transformation by ineffective A-MuLV (N35-MuLV). The A-MuLV genome is responsible for hematopoietic cell transformation because a nonproducer clone of lymphoid cells, free of helper virus, was isolated. The data indicates that the pseudotype of A-MuLV determines its ability to transform hematopoietic cells.

Thymic lymphoma induction by the AKT8 murine retrovirus.

The directly transforming murine retrovirus, AKT8, was isolated from a spontaneous AKR thymoma and carries the cell-derived viral oncogene, akt. We have now shown that this virus produces thymic lymphomas after inoculation of susceptible mouse strains. The presence of the AKT8 genome in the DNA of the virus-induced tumors was demonstrated by Southern blotting using an akt-specific probe. These results establish the in vivo pathogenicity of the AKT8 virus and its akt oncogene, and imply a potential role for the cellular akt proto-oncogene in tumor development.

Control of pathogenicity and disease specificity of a T-lymphomagenic gammaretrovirus by E-box motifs but not by an overlapping glucocorticoid response element.

Although transcription factors of the basic helix-loop-helix family have been shown to regulate enhancers of lymphomagenic gammaretroviruses through E-box motifs, the overlap of an E-box motif (Egre) with the glucocorticoid response element (GRE) has obscured their function in vivo. We report here that Egre, but not the GRE, affects disease induction by the murine T-lymphomagenic SL3-3 virus. Mutating all three copies of Egre prolonged the tumor latency period from 60 to 109 days. Further mutating an E-box motif (Ea/s) outside the enhancer prolonged the latency period to 180 days, suggesting that Ea/s works as a backup site for Egre. While wild-type SL3-3 and GRE and Ea/s mutants exclusively induced T-cell lymphomas with wild-type latencies mainly of the CD4(+) CD8(-) phenotype, Egre as well as the Egre and Ea/s mutants induced B-cell lymphomas and myeloid leukemia in addition to T-cell lymphomas. T-cell lymphomas induced by the two Egre mutants had the same phenotype as those induced by wild-type SL3-3, indicating the incomplete disruption of T-cell lymphomagenesis, which is in contrast to previous findings for a Runx site mutant of SL3-3. Mutating the Egre site or Egre and Ea/s triggered several tumor phenotype-associated secondary enhancer changes encompassing neighboring sites, none of which led to the regeneration of an E-box motif. Taken together, our results demonstrate a role for the E-box but not the GRE in T lymphomagenesis by SL3-3, unveil an inherent broader disease specificity of the virus, and strengthen the notion of selection for more potent enhancer variants of mutated viruses during tumor development.

Porcine endogenous retroviruses and xenotransplantation.

Xenotransplantation is defined by the PHS as any procedure that involves the transplantation, implantation or infusion into a human recipient of either (a) live cells, tissues or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs (Public Health Service Guideline on Infectious Disease Issues in Xenotransplantation). Use of pigs for human xenotransplantation raises concerns about the risks of transfer of infectious agents from the pig cells to xenotransplantation recipients. The observation that the porcine germline harbors genetic loci encoding porcine endogenous retroviruses (PERVs) that are in some cases infectious for human cells has resulted in renewed scientific interest in PERVs. However, in spite of the past 10 years of investigation, the actual risk for PERV infection, replication, and pathogenic outcome in human recipients of xenotransplantation products is still undefined. (Part of a multi-author review).

Adaptive evolution of a tagged chimeric gammaretrovirus: identification of novel cis-acting elements that modulate splicing.

Retroviruses are well known for their ability to incorporate envelope (Env) proteins from other retroviral strains and genera, and even from other virus families. This characteristic has been widely exploited for the generation of replication-defective retroviral vectors, including those derived from murine leukemia virus (MLV), bearing heterologous Env proteins. We investigated the possibility of "genetically pseudotyping" replication-competent MLV by replacing the native env gene in a full-length viral genome with that of another gammaretrovirus. Earlier, we developed replication-competent versions of MLV that stably transmit and express transgenes inserted into the 3' untranslated region of the viral genome. In one such tagged MLV expressing green fluorescent protein, we replaced the native env sequence with that of gibbon ape leukemia virus (GALV). Although the GALV Env protein is commonly used to make high-titer pseudotypes of MLV vectors, we found that the env replacement greatly attenuated viral replication. However, extended cultivation of cells exposed to the chimeric virus resulted in selection of mutants exhibiting rapid replication kinetics and different variants arose in different infections. Two of these variants had acquired mutations at or adjacent to the splice acceptor site, and three others had acquired dual mutations within the long terminal repeat. Analysis of the levels of unspliced and spliced viral RNA produced by the parental and adapted viruses showed that the mutations gained by each of these variants functioned to reverse an imbalance in splicing caused by the env gene substitution. Our results reveal the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and demonstrate that tagging of the retroviral genome with an easily assayed transgene can be combined with in vitro evolution as an approach to efficiently generating and screening for replicating mutants of replication-impaired recombinant viruses.

Presence of prelymphoma cells in the bone marrow of the lymphomagenic virus-treated AKR mouse.

Prelymphoma cells (PLC) are defined as cells which give rise to lymphoma cells but are not in themselves autonomous. They are present in the bone marrow of young AKR mice, a strain with a high natural incidence of thymic lymphoma. PLC are identified by transfer of AKR bone marrow into 400-rad-treated F1 recipients, one of the parents being AKR. Normal 1-month-old AKR bone marrow cells result in thymic lymphomas of AKR type in the hybrid recipients after latent periods of 6 to 16 months. In the present study, PLC resulting in lymphoma of AKR type 3 to 4 months after inoculation to irradiated hybrids are described. They are consistently found in the bone marrow of 21- to 28-day-old AKR mice treated at 3 to 5 days of age with a lymphomagenic virus, SL3-3. Long-term culture of these bone marrow cells allows the survival of totipotent hemopoietic stem cells but not of PLC. Thymic stromal remnants prepared from the 21- to 28-day-old virus-treated mice efficiently replicate oncogenic virus; however, they do not contain PLC. This was determined by grafting the remnants to irradiated and nonirradiated hybrid recipients. No AKR type lymphomas developed in the grafted mice. We conclude that the bone marrow of young oncogenic virus-treated AKR mice contains PLC modified by oncogenic virus so that they can produce thymic lymphomas after a shorter latent period than can PLC found in 1-month-old normal AKR mice. The modified PLC are not derived from totipotent marrow stem cells or sequestered in the thymic stroma of virus-treated mice.

Viruses and the placenta: the essential virus first view.

A virus first perspective is presented as an alternative hypothesis to explain the role of various endogenized retroviruses in the origin of the mammalian placenta. It is argued that virus-host persistence is a key determinant of host survival and the various ERVs involved have directly affected virus-host persistence.

Perspectives in studies of human tumor viruses.

Tumor viruses can be found in both the RNA and DNA virus kingdoms. All RNA tumor viruses belong to the retrovirus family. Directly transforming Class I RNA tumor viruses carry cellular oncogenes, picked up by accidental recombination, and usually selected for secondary modifications and high tumorigenicity by the investigator. They are not known to play any role for tumor causation in nature. Class II or chronic RNA tumor viruses do not carry cell-derived oncogenes but they often act by proviral DNA insertion into the immediate neighborhood of a cellular oncogene. Feline, murine, and avian leukemia viruses belong to this category. The human adult T-cell leukemia virus, (HTLV-1) and bovine leukemia virus (BLV) act by expanding the preneoplastic cell population and thereby provides the soil for secondary, cellular changes. The DNA tumor viruses belong to three very different categories, the papovaviruses, adenoviruses and herpesviruses. Inactivation of the Rb and the p53 pathway by the viral transforming proteins is a convergent feature of the papova- and the adenoviruses. Since all DNA tumor viruses kill their host cell following their entry into the lytic phase, transformation and tumorigenicity are entirely dependent on a non-lytic interaction. Cells transformed by DNA tumor viruses depend on the continued expression of the virally encoded oncogene. They provide thereby a convenient target for the immune surveillance of the host. Depending on the epidemiological history of the virus in relation to its natural host species, the immune surveillance of the host and the strategy of viral latency and survival can evolve into a truly symbiotic relationship, as best illustrated by the Epstein-Barr virus (EBV). Tumor development occurs only as an accident at the level of the host (immunosuppression) or the cell (specific translocations or other genetic changes). The list of human viruses presently known to cause or to contribute to tumor development comprise four DNA viruses, namely Epstein-Barr virus, certain human papilloma viruses subtypes, hepatitis B virus, and Kaposi sarcoma herpesvirus (HHV-8); and two RNA viruses, adult T-cell leukemia virus (HTLV-1) and hepatitis virus C.