Biochemical features of bovine leukemia virus
Daniel PortetelleR. KettmannM. MammerickxY. CleuterD. DekegelM. GalouxJacques GhysdaelA. BurnyHubert Chantrenne
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Keywords:
Bovine leukemia virus
Oncovirus
Gammaretrovirus
Feline leukemia virus
Endogenous retrovirus
Oncovirus
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Bovine leukemia virus
Provirus
Oncovirus
Deltaretrovirus
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Tumor Virus
Gammaretrovirus
Oncovirus
Budding
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Certain glycosaminoglycans (GAGs), including heparin, inhibit infection by murine leukemia virus (MLV). We now show that this is due to inhibition of virus attachment independent of the interaction between viral envelope proteins (Env) and their cellular receptors. Heparin blocked the binding of both Env-deficient and amphotropic MLV (MLV-A) particles to NIH 3T3 fibroblasts, CHO cells which lack the amphotropic retroviral receptor Pit-2, and CHO cells transfected with Pit-2 (CHO-Pit-2). Heparin also inhibited the transduction of NIH 3T3 cells by MLV-A over a similar concentration range. This effect was observed within 15 min of exposure to retrovirus. Preloading target cells with heparin had no effect on transduction and both MLV-A and Env-deficient retrovirus bound efficiently to heparin-coated agarose beads, suggesting that heparin interacts with the virus rather than the target cell. This requires both a strong negative charge and a specific structure since GAGs with different charge and carbohydrate composition inhibited virus infection variably. The specificity of GAG-virus interaction also depends on the producer cells, since virus packaged by murine GP+EnvAM12 cells was 1,000-fold more sensitive to inhibition by chondroitin sulfate A than was virus packaged by human FLYA13 packaging cells. No evidence for an interaction between MLV and cell surface proteoglycans was found, however, since the attachment of MLV-A and envelope-defective virus to proteoglycan-deficient CHOpgsA-745 cells was similar to that seen with both wild-type and CHO-Pit-2 cells. Although the molecular mechanism is unclear, this study presents evidence that Env receptor-independent attachment is an important step in MLV infection.
Gammaretrovirus
3T3 cells
Helper virus
Viral transformation
Transduction (biophysics)
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ABSTRACT In the absence of envelope gene expression, retrovirus packaging cell lines expressing Moloney murine leukemia virus (MLV) gag and pol genes produce large amounts of noninfectious virus-like particles that contain reverse transcriptase, processed Gag protein, and viral RNA ( gag-pol RNA particles). We demonstrate that these particles can be made infectious in an in vitro, cell-free system by the addition of a surrogate envelope protein, the G spike glycoprotein of vesicular stomatitis virus (VSV-G). The appearance of infectivity is accompanied by physical association of the G protein with the immature, noninfectious virus particles. Similarly, exposure in vitro of wild-type VSV-G to a fusion-defective pseudotyped virus containing a mutant VSV-G markedly increases the infectivity of the virus to titers similar to those of conventional VSV-G pseudotyped viruses. Furthermore, similar treatment of an amphotropic murine leukemia virus significantly allows infection of BHK cells not otherwise susceptible to infection with native amphotropic virus. The partially cell-free virus maturation system reported here should be useful for studies aimed at the preparation of tissue-targeted retrovirus vectors and will also aid in studies of nucleocapsid-envelope interactions during budding and of virus assembly and virus-receptor interactions during virus uptake into infected cells. It may also represent a potentially useful step toward the eventual development of a completely cell-free retrovirus assembly system.
Infectivity
Group-specific antigen
Gammaretrovirus
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Transduction (biophysics)
Endogenous retrovirus
Gammaretrovirus
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Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus found in association with human prostate cancer and chronic fatigue syndrome, although these associations are controversial. XMRV shows at most 94% identity to known mouse retroviruses. Here we used XMRV-specific PCR to search for a more closely related source of XMRV in mice. While we could not find a complete copy, we did find a 3,600-bp region of XMRV in an endogenous retrovirus present in NIH/3T3 cells. These results show that XMRV has clear ancestors in mice and highlight another possible source of contamination in PCR assays for XMRV.
Gammaretrovirus
Endogenous retrovirus
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ABSTRACT Human APOBEC3G (huAPOBEC3G), also known as CEM15, is a broad antiretroviral host factor that deaminates dC to dU in the minus strand DNA of human immunodeficiency virus type 1 (HIV-1), other lentiviruses, and murine leukemia virus (MLV), thereby creating G-to-A hypermutation in the plus strand DNA to inhibit the infectivity of these viruses. In this study, we examined the antiretroviral function of a murine homologue of APOBEC3G (muAPOBEC3G) on several retrovirus systems with different producer cells. MuAPOBEC3G did not suppress the infectivity of murine retroviral vectors produced from human or murine cells, whereas it showed antiviral activity on both wild-type and Δvif virions of HIV-1 in human cells. In contrast, huAPOBEC3G showed broad antiviral activity on HIV-1 and murine retroviral vectors produced from human cells as well as murine cells. These data suggested that muAPOBEC3G does not possess antiretroviral activity on murine retroviruses and has a different target specificity from that of huAPOBEC3G and that huAPOBEC3G works as a broad antiviral factor not only in human cells but also in murine cells. A functional interaction study between human and murine APOBEC3G supported the former hypothesis. Furthermore, studies on the expression of APOBEC3G in producer cells and its incorporation into virions revealed that muAPOBEC3G is incorporated into HIV-1 virions but not into MLV virions. Thus, muAPOBEC3G cannot suppress the infectivity of murine retrovirus because it is not incorporated into virions. We suggest that murine retroviruses can replicate in murine target cells expressing muAPOBEC3G because they are not targets for this enzyme.
APOBEC3G
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Gammaretrovirus
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Retrovirus entry into cells is mediated by specific interactions between the retrovirally encoded Env envelope glycoprotein and a host cell surface receptor. Though a number of peptide motifs responsible for the structure as well as for the binding and fusion activities of Env have been identified, only a few quantitative data concerning the infection process are available. Using an inducible expression system, we have expressed various amounts of ecotropic and amphotropic Env at the surfaces of Moloney murine leukemia virus-derived vectors and assayed for the infectivity of viral particles. Contrary to the current view that numerous noncooperative Env-viral receptor interactions are required for cell infection, we report here that very small amounts of Env are sufficient for optimal infection. However, increasing Env density clearly accelerates the rate at which infectious attachment to cells occurs. Moreover, our data also show that a surprisingly small number of Env molecules are sufficient to drive infection, albeit at a reduced efficiency, and that, under conditions of low expression, Env molecules act cooperatively. These observations have important consequences for our understanding of natural retroviral infection as well as for the design of cell-targeted infection techniques involving retroviral vectors.
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Gammaretrovirus
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ABSTRACT Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in unwanted events, such as the formation of recombinant viruses and the mobilization of therapeutic vectors. Using sensitive reverse transcriptase PCR assays, we investigated human and murine gene therapy packaging cell lines for incorporation of endogenous retrovirus transcripts into murine leukemia virus (MLV) vector particles and, conversely, whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were efficiently packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived β-galactosidase (β-Gal) vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed several classes of HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Nonspecific packaging of the MLV Gag-Pol expression vector transcripts was detected in the FLY virions at a low level (1 in 17,000 sequences). These findings indicate that human packaging cells produce retrovirus particles far less contaminated by endogenous viral sequences than murine packaging cells. Human teratocarcinoma cells (GH cells), which produce HERV-K particles, were transduced with an MLV-derived β-Gal vector. Although both HERV-K and RTVL-H sequences were found in association with the particles, β-Gal transcripts were not detected, indicating that HERV Gag proteins do not efficiently package MLV-based vectors.
Endogenous retrovirus
HEK 293 cells
Gammaretrovirus
Group-specific antigen
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