Dynein Light-Chain Dynlrb2 Is Essential for Murine Leukemia Virus Traffic and Nuclear Entry.

Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.

Construction of a replication-competent retroviral vector for expression of the VSV-G envelope glycoprotein for cancer gene therapy.

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.

Resurrection of endogenous retroviruses in antibody-deficient mice.

The mammalian host has developed a long-standing symbiotic relationship with a considerable number of microbial species. These include the microbiota on environmental surfaces, such as the respiratory and gastrointestinal tracts, and also endogenous retroviruses (ERVs), comprising a substantial fraction of the mammalian genome. The long-term consequences for the host of interactions with these microbial species can range from mutualism to parasitism and are not always completely understood. The potential effect of one microbial symbiont on another is even less clear. Here we study the control of ERVs in the commonly used C57BL/6 (B6) mouse strain, which lacks endogenous murine leukaemia viruses (MLVs) able to replicate in murine cells. We demonstrate the spontaneous emergence of fully infectious ecotropic MLV in B6 mice with a range of distinct immune deficiencies affecting antibody production. These recombinant retroviruses establish infection of immunodeficient mouse colonies, and ultimately result in retrovirus-induced lymphomas. Notably, ERV activation in immunodeficient mice is prevented in husbandry conditions associated with reduced or absent intestinal microbiota. Our results shed light onto a previously unappreciated role for immunity in the control of ERVs and provide a potential mechanistic link between immune activation by microbial triggers and a range of pathologies associated with ERVs, including cancer.

Embryonic stem cells use ZFP809 to silence retroviral DNAs.

Embryonic stem cells (ESCs) and other primitive stem cells of mice have been known for more than 30 years to potently block retrovirus replication. Infection of ESCs by the murine leukaemia viruses (MLVs) results in the normal establishment of integrated proviral DNA, but this DNA is then transcriptionally silenced, preventing further viral spread. The repression is largely mediated by trans-acting factors that recognize a conserved sequence element termed the primer binding site, an 18-base pair sequence complementary to the 3' end of a cellular transfer RNA. A specific tRNA is annealed to the primer binding site sequence of the viral genomic RNA, and is used to prime DNA synthesis. This same sequence in the context of the integrated proviral DNA is targeted for silencing in ESCs. We have recently shown that a large protein complex binding to the primer binding site in ESCs contains TRIM28 (refs 8, 9), a well-characterized transcriptional co-repressor. An important question remains as to the identity of the factor that directly recognizes integrated retroviral DNAs and recruits TRIM28 to mediate their specific silencing. Here we identify the zinc finger protein ZFP809 as the recognition molecule that bridges the integrated proviral DNA and TRIM28. We show that expression of ZFP809 is sufficient to render even differentiated cells highly resistant to MLV infection. Furthermore, we demonstrate that ZFP809 is able to potently block transcription from DNA constructs of human T-cell lymphotropic virus-1 (HTLV-1), which use the same primer tRNA. These results identify ZFP809 as a DNA-binding factor that specifically recognizes a large subset of mammalian retroviruses and retroelements, targeting them for transcriptional silencing. We propose that ZFP809 evolved as a stem-cell-specific retroviral restriction factor, and therefore constitutes a new component of the intrinsic immune system of stem cells.

Profound amplification of pathogenic murine polytropic retrovirus release from coinfected cells.

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98-F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.

TANK-binding kinase 1 attenuates PTAP-dependent retroviral budding through targeting endosomal sorting complex required for transport-I.

Retroviruses need to bud from producer cells to spread infection. To facilitate its budding, some virus hijacks the multivesicular body (MVB) pathway that is normally used to cargo and degrade ubiquitylated cellular proteins, through interaction between the late domain of Gag polyproteins and the components of MVB machinery. In this study, we demonstrated that TANK-binding kinase 1 (TBK1) directly interacted with VPS37C, a subunit of endosomal sorting complex required for transport-I (ESCRT-I) in the MVB pathway, without affecting the ultrastructure or general function of MVB. Interestingly, overexpression of TBK1 attenuated, whereas short hairpin RNA interference of TBK1 enhanced HIV-1 pseudovirus release from Vero cells in type I IFN (IFN-I)-independent manner. Down-regulation of TBK1 by short hairpin RNA in TZM-bl cells also enhanced live HIV-1 NL4-3 or JR-CSF virus budding without involvement of IFN-I induction. Furthermore, infection of TBK1-deficient mouse embryonic fibroblast cells with a chimeric murine leukemia virus/p6, whose PPPY motif was replaced by PTAP motif of HIV-1, showed that lack of TBK1 significantly enhanced PTAP-dependent, but not PPPY-dependent retrovirus budding. Finally, phosphorylation of VPS37C by TBK1 might regulate the viral budding efficiency, because overexpression of the kinase-inactive mutant of TBK1 (TBK1-K38A) in Vero cells accelerated HIV-1 pseudovirus budding. Therefore, through tethering to VPS37C of the ESCRT-I complex, TBK1 controlled the speed of PTAP-dependent retroviral budding through phosphorylation of VPS37C, which would serve as a novel mechanism of host cell defense independent of IFN-I signaling.

Molecular breeding of viruses.

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein.

BACKGROUND: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. RESULTS: A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 µM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway. CONCLUSIONS: The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.

The absence of IDO upregulates type I IFN production, resulting in suppression of viral replication in the retrovirus-infected mouse.

Indoleamine 2,3-dioxygenase, the L-tryptophan-degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO(-/-)) and 1-methyl-D-L-tryptophan-treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO(-/-) mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO(-/-) mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO(-/-) mice or 1-methyl-D-L-tryptophan-treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO(-/-) mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

The receptors for gibbon ape leukemia virus and amphotropic murine leukemia virus are not downregulated in productively infected cells.

BACKGROUND: Over the last several decades it has been noted, using a variety of different methods, that cells infected by a specific gammaretrovirus are resistant to infection by other retroviruses that employ the same receptor; a phenomenon termed receptor interference. Receptor masking is thought to provide an earlier means of blocking superinfection, whereas receptor down regulation is generally considered to occur in chronically infected cells. RESULTS: We used replication-competent GFP-expressing viruses containing either an amphotropic murine leukemia virus (A-MLV) or the gibbon ape leukemia virus (GALV) envelope. We also constructed similar viruses containing fluorescence-labeled Gag proteins for the detection of viral particles. Using this repertoire of reagents together with a wide range of antibodies, we were able to determine the presence and availability of viral receptors, and detect viral envelope proteins and particles presence on the cell surface of chronically infected cells. CONCLUSIONS: A-MLV or GALV receptors remain on the surface of chronically infected cells and are detectable by respective antibodies, indicating that these receptors are not downregulated in these infected cells as previously proposed. We were also able to detect viral envelope proteins on the infected cell surface and infected cells are unable to bind soluble A-MLV or GALV envelopes indicating that receptor binding sites are masked by endogenously expressed A-MLV or GALV viral envelope. However, receptor masking does not completely prevent A-MLV or GALV superinfection.

Expression of endogenous murine leukemia viruses during the course of a protracted immunological disorder.

Mice of the low leukemia (BALB/cJ x A/J)F1 hybrid (CAF1) strain express B-and N-tropic infectious murine leukemia virus (MuLV) after the age of 6 mo. Initation of a protracted immunological disorder, the graft-versus-host reaction (GVHR), at 7 wk of age, accelerates the induction of both these mouse-tropic endogenous viruses, and preferentially enhances the replication of B-tropic MuLV. The earlier appearance of B-tropic MuLV in a greater proportion of mice and in higher titer is thought to be casually related to the eventual development of lymphoreticular tumors in the GVHR mice, since previous studies have shown that these same tumors can be reproduced by inoculating syngeneic recipients with serially passaged GVHR extracts containing B-tropic MuLV.

Expression of xenotropic murine leukemia viruses as cell-surface gp70 in genetic crosses between strains DBA/2 and C57BL/6.

Flow microfluorometry was used to assess levels of xenotropic murine leukemia virus envelope-related cell-surface antigens (XenCSA) expressed on lymphocytes of mice derived from crosses between C57BL/6 (B6) and DBA/2 (D2); 24 recombinant inbred strains (BXD RIs) and 62 backcross mice were studied. The results suggest that XenCSA expression is affected by more than one gene but that the predominant influence is exerted by a single semidominant gene apparently located on chromosome 4 at or in close proximity to the Fv-1 locus. Studies of spontaneous virus production in B6D2F1 X D2 mice suggest that this locus may also affect production by spleen cells of xenotropic MuLV registering in a fluorescent antibody assay of mink lung cells.

Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: pleiotropic restriction of endogenous BALB virus in cat X mouse somatic cell hybrids.

Bvr-1 is a dominant X-linked feline gene which restricts the replication of B-tropic murineleukemia virus (B-MuLV) in somatic cell hybrids between murine BALB/c-RAG cells and FL-74 feline cells. Since the hybrids were originally derived by the hypoxanthine aminopterin thymidine selection scheme, counter selection experiments on 6-thioguanine result in preferential survival of hybrid cells which have spontaneously lost the feline X-chromosome on which is located the structural gene for hypoxanthine guanine phosphoribosyl transferase (IMP: pyrophosphate phosphoribosyl transferase, E.C. 2.4.2.8) and Bvr-1. Back selected Bvr-1- cells express high parental levels of B-MuLV. Bvr-1 effectively restricts the IdU-mediated induction of the endogenous xenotropic BALB virus (BALB: virus 2) but not the endogenous N-tropic virus (BALB: virus 1). Pleiotropic restriction of B-MuLV and X-MuLV, but not N-MuLV suggests that the viral targets of Bvr-1 (either viral components or functions in viral assembly) of the B-tropic and X-tropic endogenous BALB viruses are similar to each other but distinct from the target in the N-tropic virus. Very low levels of B-MuLV are detected in restricted cells, but this residual virus is not infectious in either NIH-3T3 or BALB-3T3 mouse cells which are genotypically Fv-1N/Fv-1N and Fv-1B/Fv-1B, respectively. Passage of residual virus through host cells without Fv-1 related restriction (SC-1) results in production of infectious B-MuLV indistinguishable from that produced by RAG parent cells.

Allelism and linkage studies of murine leukemia virus activation genes in low leukemic strains of mice.

Previously, we identified two genes, termed Inc-1 and Inb-1, that interact to enhance ecotropic murine leukemia virus induction in low virus strains of mice. Mice related to BALB/c in origin carry a locus termed Inc-1, whereas mice related to B6 carry an Inb-1 locus. Mice that carry both Inc-1 and Inb-1 yield 10- to 50-fold more virus-producing cells than parental strains on induction with halogenated pyrimidines in vitro and demonstrate enhanced murine leukemia virus production in vivo. Here, we show that mice related to BALB/c in origin, i.e., A, C3H/He, and SEC, have an Inc-1 locus that is allelic with that of BALB/c. The C57BR mouse strain has an Inb-1 locus that is allelic with that of B6, located on chromosome 8, 30 cM from Es-1. We also show that the Inc-1 locus of BALB/c mice is located on chromosome 5, 24 cM from Pgm-1 and 43 cM from Gus. Kozak and Rowe (6,8) and Ihle and co-workers (3) have shown that the ecotropic virus-inducing genes in BALB/c and B10 mice are located on chromosomes 5 and 8, respectively, with similar distances from the previously mentioned biochemical markers. Our data are consistent with two possibilities: Inc-1 and Inb-1 are part of the virus-inducing genes Cv-1 and Bv-1, respectively , or Inc-1 and Inb2-1 are tightly linked regulatory genes.

Genetic factors influencing C-type RNA virus induction.

Genetic factors were studied which affect the inducibility of C-type RNA viruses from embyro cultures of mouse strains with high and low incidence of spontaneous leukemia. Virus was inducible by chemical treatment from secondary embryo cultures of several inbred strains. Both virus inducibility and the capacity of the virus to persist in cells from which it was activated were found to be inherited as dominant genetic characteristics. The results show that the factors controlling virus activation and persistence in culture also play an important role in spontaneous virus expression in the animal.

Suppression of endogenous murine leukemia virus by maternal resistance factor.

Females of the RF and SJL inbred mouse strains transmit to their progeny of both sexes a nonmendelian maternal resistance factor (MRF) able to suppress the expression of endogenous ecotropic murine leukemia virus (E-MuLV). This MRF is demonstrable in crosses with AKR mice by comparing E-MuLV expression in the spleens and thymuses of reciprocal F1 generations. DBA/2 and ST/b mice are MRF negative by these criteria. Neonatal inoculation of E-MuLV-containing spleen extracts gives rise to persistent expression of infectious virus in mice of the MRF- but not the MRF+ strains. However, inoculation of the virus in 30-d-old females of the MRF- strains no longer leads to a state of persistent infection; instead, these females become MRF+ and transmit protection against E-MuLV expression to their progeny by AKR and RF males. The MRF appears to be transmitted to the progeny mainly through the milk, since foster-nursing AKR neonates on RF (but not DBA/2) mothers greatly reduces E-MuLV expression in the progeny. These RF-fostered AKR mice also show a reduced and delayed lymphoma incidence, a finding consistent with the idea that maternally transmitted resistance to E-MuLV expression is the basis for the classic maternal resistance to lymphomagenesis seen in the progeny of RF mothers.

Tumor induction by immunologically activated murine leukemia virus.

A graft-vs.-host reaction (GVHR) was induced in young male CAF(I) and CB6F(1) mice by the administration of BALB/cJ spleen cells. A proportion of such mice subsequently developed lymphoreticular rumors. Cell-free extracts (CFEs) prepared from the reticular tissues of CAF(1) mice killed at intervals after the induction of the GVHR were tested for their capacity to produce the same tumors in a litter of syngeneic mice inoculated at birth. 12 of 29 (41.4%) such extracts were positive, causing lymphoreticular tumors in one or more littermate recipients. The positive CFEs came from donors killed at all stages of the GVHR, from tumor-bearing mice as well as from non-tumor-bearing mice. However, whereas less than 30% of CFEs from mice killed within 12 mo of GVHR induction were oncogenic, the incidence of oncogenic extracts from mice killed 12-15 mo after GVHR induction rose to 75%. None of the CFEs prepared from nine normal uninjected male CAF(1) mice killed between the ages of 8 and 18 mo transmitted tumors to recipients. CFEs prepared from CAF(1) mice with the GVHR were tested for infectious murine leukemia virus (MuLV) using the XC assay and also for complement-fixing (CF) group-specific MuLV antigen. Substantial titers of B-tropic MuLV and CF antigen were detected in at least half the extracts from mice killed 11-14 mo after GVHR induction. During the first few months of GVHR induction, MuLV titers were low and CF antigen was absent. Neither infectious MuLV nor CF antigen were detected in CFEs prepared from normal control mice. Serially passed CFEs originating from a CB6F(1) GVHR-induced RCN caused similar tumors in successive generations of syngeneic recipient mice. These lymphoreticular tumors were shown to contain infectious MuLV, CF MuLV antigen, and C-type particles. These data together provide evidence that MuLV is activated during the GVHR and that it is responsible for the eventual development of lymphoreticular tumors.

Mobilization of endogenous retroviruses in mice after infection with an exogenous retrovirus.

Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.

Unintended spread of a biosafety level 2 recombinant retrovirus.

BACKGROUND: Contamination of vertebrate cell lines with animal retroviruses has been documented repeatedly before. Although such viral contaminants can be easily identified with high sensitivity by PCR, it is impossible to screen for all potential contaminants. Therefore, we explored two novel methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant. RESULTS: The first hint for the presence of contaminating retroviruses in one of our cell lines was obtained by electron microscopy of exosome-like vesicles released from the supernatants of transfected 293T cells. Random amplification of particle associated RNAs (PAN-PCR) from supernatant of contaminated 293T cells and sequencing of the amplicons revealed several nucleotide sequences showing highest similarity to either murine leukemia virus (MuLV) or squirrel monkey retrovirus (SMRV). Subsequent mass spectrometry analysis confirmed our findings, since we could identify several peptide sequences originating from monkey and murine retroviral proteins. Quantitative PCRs were established for both viruses to test currently cultured cell lines as well as liquid nitrogen frozen cell stocks. Gene fragments for both viruses could be detected in a broad range of permissive cell lines from multiple species. Furthermore, experimental infections of cells negative for these viruses showed that both viruses replicate rapidly to high loads. We decided to further analyze the genomic sequence of the MuLV-like contaminant virus. Surprisingly it was neither identical to MuLV nor to the novel xenotropic MuLV related retrovirus (XMRV) but showed 99% identity to a synthetic retrovirus which was engineered in the 1980s. CONCLUSION: The high degree of nucleotide identity suggests unintended spread of a biosafety level 2 recombinant virus, which could also affect the risk assessment of gene-modified organisms released from contaminated cell cultures. The study further indicates that both mass spectrometry and PAN-PCR are powerful methods to identify viral contaminations in cell lines without prior knowledge of the kind of contaminant. Both methods might be useful tools for testing cell lines before using them for critical purposes.

Interfering residues narrow the spectrum of MLV restriction by human TRIM5alpha.

TRIM5alpha is a restriction factor that limits infection of human cells by so-called N- but not B- or NB-tropic strains of murine leukemia virus (MLV). Here, we performed a mutation-based functional analysis of TRIM5alpha-mediated MLV restriction. Our results reveal that changes at tyrosine(336) of human TRIM5alpha, within the variable region 1 of its C-terminal PRYSPRY domain, can expand its activity to B-MLV and to the NB-tropic Moloney MLV. Conversely, we demonstrate that the escape of MLV from restriction by wild-type or mutant forms of huTRIM5alpha can be achieved through interdependent changes at positions 82, 109, 110, and 117 of the viral capsid. Together, our results support a model in which TRIM5alpha-mediated retroviral restriction results from the direct binding of the antiviral PRYSPRY domain to the viral capsid, and can be prevented by interferences exerted by critical residues on either one of these two partners.