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• W2113203969 abstract "Article3 February 2003free access Restriction of multiple divergent retroviruses by Lv1 and Ref1 Theodora Hatziioannou Theodora Hatziioannou Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA Search for more papers by this author Simone Cowan Simone Cowan Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA Search for more papers by this author Stephen P. Goff Stephen P. Goff Department of Biochemistry and Molecular Biophysics, and Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY, 10032 USA Search for more papers by this author Paul D. Bieniasz Paul D. Bieniasz Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA Search for more papers by this author Greg J. Towers Corresponding Author Greg J. Towers Department of Biochemistry and Molecular Biophysics, and Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY, 10032 USA Wohl Virion Centre, Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London, W1T 4JF UK Search for more papers by this author Theodora Hatziioannou Theodora Hatziioannou Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA Search for more papers by this author Simone Cowan Simone Cowan Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA Search for more papers by this author Stephen P. Goff Stephen P. Goff Department of Biochemistry and Molecular Biophysics, and Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY, 10032 USA Search for more papers by this author Paul D. Bieniasz Paul D. Bieniasz Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA Search for more papers by this author Greg J. Towers Corresponding Author Greg J. Towers Department of Biochemistry and Molecular Biophysics, and Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY, 10032 USA Wohl Virion Centre, Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London, W1T 4JF UK Search for more papers by this author Author Information Theodora Hatziioannou1, Simone Cowan1, Stephen P. Goff2, Paul D. Bieniasz1 and Greg J. Towers 2,3 1Aaron Diamond AIDS Research Center and the Rockefeller University, 455 First Avenue, New York, NY, 10016 USA 2Department of Biochemistry and Molecular Biophysics, and Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY, 10032 USA 3Wohl Virion Centre, Department of Immunology and Molecular Pathology, University College London, 46 Cleveland Street, London, W1T 4JF UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:385-394https://doi.org/10.1093/emboj/cdg042 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The mouse gene Fv1 encodes a saturable restriction factor that selectively blocks infection by N-tropic or B-tropic murine leukemia virus (MLV) strains. Despite the absence of an Fv1 gene, a similar activity is present in humans that blocks N-MLV infection (Ref1). Moreover, some non-human primate cell lines express a potentially related inhibitor of HIV-1 and/or SIVmac infection (Lv1). Here, we examine the spectrum of retrovirus-restricting activities expressed by human and African green monkey cell lines. Human cells restrict N-MLV and equine infectious anemia virus (EIAV), but not HIV-1, HIV-2, SIVmac or SIVagm, whilst AGM cells restrict N-MLV, EIAV, HIV-1, HIV-2 and SIVmac. Remarkably, in each example examined, restriction of infection by a given retrovirus can be abrogated at least partially by saturation with another retrovirus, provided that it is also restricted but regardless of whether it is closely related. These data suggest that restriction factors in human and non-human primate cells are able to recognize and block infection by multiple, widely divergent retroviruses and that the factors themselves may be related. Introduction Retroviruses are dependent on a variety of host cell functions for the completion of their life cycle. As such, retroviral tropism is determined primarily by whether components of a given retrovirus are able to exploit each of the host cell factors required for viral replication. However, retroviral tropism can also be restricted by the presence of dominant-acting inhibitory activities, expressed by target cells, that prevent the efficient progression of particular steps in the viral life cycle. Presumably, these cellular inhibitors have arisen as an evolutionary consequence of pathogenic retrovirus epidemics. An example is the recently identified cellular protein, CEM15, that acts in human immunodeficiency virus type 1 (HIV-1)-producing cells to attenuate the infectivity of progeny virions. The inhibitory effect of CEM15 is counteracted by the viral Vif protein (Sheehy et al., 2002). A distinct class of inhibitory activities is exemplified by the Fv1-mediated restriction of murine leukemia virus (MLV) infection (reviewed in Jolicoeur, 1979; Goff, 1996). Fv1 was first described as a dominant, heritable trait in inbred mouse strains that confers resistance to infection by particular MLV strains (Lilly, 1970; Pincus et al., 1971). One of the two major alleles of Fv1, Fv1n, permits infection by N-tropic but restricts B-tropic MLV strains, while the other, Fv1b, permits infection of B-tropic infection but restricts N-tropic infection. At low levels of infection, the resistance conferred by Fv1 can be substantial, but is overcome by inoculating cells with high titers of restricted virions (Decleve et al., 1975; Tennant et al., 1979; Boone et al., 1990). Infection is blocked in restricting cells at a post-entry step, after substantial levels of reverse transcription have occurred, but before proviral integration (Jolicoeur and Baltimore, 1976; Pryciak and Varmus, 1992). The precise mechanism of action of Fv1 is unknown, but the viral determinants that confer sensitivity to restriction have been mapped to the capsid (CA) domain of MLV Gag (DesGroseillers and Jolicoeur, 1983), and a single amino acid substitution, arginine to glutamate at position CA110, is sufficient to convert an N-tropic MLV strain to B-tropism, and vice versa (Kozak and Chakraborti, 1996). The Fv1 gene has been identified by positional cloning, and encodes a protein with ∼60% homology to the Gag proteins encoded by the HERV-L and MERV-L families of endogenous retroviruses (Best et al., 1996; Benit et al., 1997). Based in part on the specific restriction of N- and B-MLV strains by reciprocal Fv1 variants, the simplest model for restriction involves direct recognition of the capsid of incoming virions by the Fv1 protein (Goff, 1996; Stoye, 1998; Bishop et al., 2001), but demonstration of a direct interaction has proved elusive. Non-murine mammalian cells do not encode a gene with strong sequence homology to Fv1 (Best et al., 1996). Nevertheless, cells from several mammalian species, including humans, are able specifically to restrict N-tropic MLV (Towers et al., 2000, 2002). Remarkably, the same single amino acid within CA that differentiates between N- and B-tropism in mice also determines tropism for human cells. Because of this, and other similarities to Fv1-mediated restriction (see below), non-murine cells are assumed to express a factor, termed Ref1 in humans, that restricts MLV infection. In fact, the only obvious difference in the phenotypes conferred by Fv1b and Ref1 is that Fv1 acts primarily after the completion of reverse transcription, while Ref1 prevents the accumulation of reverse transcripts (Jolicoeur and Baltimore, 1976; Pryciak and Varmus, 1992; Towers et al., 2000). Post-entry, pre-integration restrictions to retroviral tropism are not confined to MLV. HIV-1 infection of several non-human primates, for example macaques, is blocked at an early step (Shibata et al., 1995; Himathongkham and Luciw, 1996), even though the HIV-1 envelope protein is fully able to support the replication of chimeric viruses containing SIVmac Gag-Pol proteins in macaque cells (Shibata et al., 1991; Li et al., 1992, 1995; Luciw et al., 1995; Joag et al., 1996). In addition, the innate resistance of primate cells to either HIV-1 or SIVmac infection is observed even when virions are pseudotyped with the pan-tropic vesicular stomatitis virus envelope glycoprotein (VSV-G) (Hofmann et al., 1999). We have recently presented compelling evidence that the post-entry resistance of primate cells to infection by HIV-1 and SIVmac is due to the presence of an Fv1-like inhibitor (Besnier et al., 2002; Cowan et al., 2002). The resistant phenotype is dominant in human–monkey heterokaryons, is independent of the route of virus entry and acts prior to the completion of reverse transcription. Moreover, we have also demonstrated that the differential susceptibility of HIV-1 and SIVmac to restriction in some primate cell lines is determined by sequences within the CA-p2 region of Gag (Cowan et al., 2002). The restriction factor whose existence in non-human primates is inferred by these findings is termed Lv1. One of the shared hallmarks of Fv1-, Ref1- and Lv1-mediated resistance to retroviral infection is that the block is most evident at a low multiplicity of infection (m.o.i.), and that infection is cooperative at a high m.o.i. (Decleve et al., 1975; Duran-Troise et al., 1977; Tennant et al., 1979; Besnier et al., 2002; Cowan et al., 2002; Towers et al., 2002). Where restriction is potent (≥20-fold), pronounced non-linearity, or multi-hit kinetics, are evident in viral titration curves. In other words, infection by an otherwise restricted virus is strongly facilitated by the presence of other restricted virion particles. This ‘abrogation’ of restriction by high doses of restricted virions is consistent with the presence of a saturable restriction factor and not with the lack of an essential activity in restricting target cells. Furthermore, the abrogating virus must itself be restricted, and recognized by the restriction factor, in order to saturate it and facilitate infection. For example, treatment of target cells with high levels of N-MLV abrogates Fv1b- or Ref1-mediated restriction of N-MLV reporter virus infection, whilst treatment with unrestricted B-MLV does not (Boone et al., 1990; Towers et al., 2002). We have previously used abrogation and other assays to demonstrate that HIV-1 and SIVmac are differentially restricted in cell lines from various primate species (Besnier et al., 2002; Cowan et al., 2002). In this study, we have found that some human and non-human primate cell lines can restrict diverse retroviruses, and that in each case examined, restriction can be abrogated by a second retrovirus, provided that it is also restricted in the same cell line. Abrogation of restriction to one retrovirus by another does not appear to depend on the sequence homology between the two viruses. These data indicate that restriction factors in primates are capable of targeting multiple, widely divergent retroviruses. Results Restriction of N-MLV in primate cells It has been shown previously that N-MLV is restricted by an Fv1-like activity in human and African green monkey (AGM) cells (Towers et al., 2000). Furthermore lentivirus infection is restricted in Old World (rhesus and African green) as well as New World (owl and squirrel) monkey cell lines (Besnier et al., 2002; Cowan et al., 2002). To determine whether the resistance to primate lentiviruses correlates with resistance to MLVs, we examined the distribution of N- or B-MLV restricting factors in primates. N-green fluorescent protein (GFP) and B-GFP vectors were titrated on a series of cell lines from primate species that we have shown previously to restrict infection by HIV-1, SIVmac or both. As is shown in Figure 1, three AGM cell lines exhibit a Ref1-like phenotype, in that they were less susceptible (>100-fold) to infection by N-GFP than by B-GFP. Moreover, at high inoculum, the titration curves of N-MLV revealed multi-hit kinetics of infection. In contrast, rhesus and owl monkey cell lines, which restrict HIV-1, did not restrict either N-GFP or B-GFP detectably. A squirrel monkey cell line that is highly resistant to SIVmac was marginally more susceptible to B-GFP than to N-GFP, but this difference was only ∼2-fold. Figure 1.Restriction of N-MLV in AGM cell lines but not those of other primates that restrict HIV-1 or SIVmac. Each primate cell line was infected with serially diluted N-GFP or B-GFP vectors. Each vector stock was also titrated on Fv1-null MDTF cells, and the inoculum level is given in MDTF infectious units (i.u.). The percentage of infected (GFP-positive) cells is plotted for each level of N-GFP or B-GFP inoculum. Download figure Download PowerPoint Abrogation of restriction to MLV infection in AGM cells by primate lentiviruses In order to investigate the relationship between factors that restrict infection by MLV and lentiviruses, we performed a series of experiments in AGM cells, which restrict both. Because restriction factors are saturable, treatment of target cells with high levels of a restricted virus can abrogate the restriction and facilitate infection by a second restricted virus. This type of experiment is shown in Figure 2A, where treatment of AGM CV-1 cells with increasing amounts of HIV-2-Puro particles can enhance infection by a fixed inoculum of a homologous HIV-2-GFP vector, in a dose-dependent manner. In contrast, HIV-2-GFP infection is not increased in non-restricting human TE671 cells by excess HIV-2 particles. This strategy can also be used to test whether factors that restrict a particular virus can be saturated by a heterologous virus, as we have shown previously for HIV-1 and SIVmac in AGM and rabbit cells (Besnier et al., 2002; Cowan et al., 2002). As is shown in Figure 2B, incubation of CV-1 target cells with increasing amounts of HIV-2 particles enhanced infection by the SIV-GFP vector by >10-fold. These data suggest that a single form of Lv1 in CV-1 cells restricts and can be saturated by HIV-1, HIV-2 and SIVmac. Figure 2.Saturable resistance to primate lentiviruses and N-MLV in CV-1 cells. (A) HIV-2 is restricted in CV-1 (AGM) but not in human (TE671) cells. CV-1 cells (filled symbols) or TE671 cells (open symbols) were inoculated with a fixed dose of HIV-2-GFP vector in the presence of increasing amounts of abrogating HIV-2-Puro particles. The percentage of HIV-2-GFP-infected cells as a function of abrogating virus dose is plotted. Abrogating virus dose is given in infectious units (i.u.) measured as puromycin-resistant colony formation on TE671 cells. (B) Abrogation of SIV-GFP restriction by HIV-2-Puro particles. CV-1 cells were inoculated with a fixed dose of SIV-GFP in the presence of abrogating HIV-2 particles, as in (A), and the percentage of SIV-GFP infected cells is plotted. (C) Resistance of CV-1 cell to N-GFP is saturable. CV-1 cells were inoculated with a fixed dose of N-GFP in the presence of increasing levels of abrogating N-Neo or B-Neo vector particles. Abrogating virus dose is given as MDTF i.u. as measured by G418-resistant colony formation. Download figure Download PowerPoint We used the same abrogation of restriction strategy to confirm that the resistance of AGM cells to N-MLV infection is saturable, as has been shown previously in human cells (Towers et al., 2002). As expected, and shown in Figure 2C, treatment of CV-1 target cells with N-Neo vector dramatically facilitated infection by an N-GFP vector. This abrogation was specific, because B-Neo particles had no abrogating effect. Thus, like human cells, this AGM cell line presumably harbors a restriction factor with properties similar to those of Ref1 and Fv1b, i.e. it can be saturated by N-MLV but not B-MLV particles. Similar results were obtained in a second AGM cell line (Vero; data not shown). Next we examined whether primate lentivirus particles could abrogate the restriction of N-MLV that is evident in AGM cells. CV-1 target cells were treated with HIV-1, HIV-2 or SIVmac particles containing a packageable vector genome encoding Puro or LacZ. Remarkably, and as shown in Figure 3A, increasing amounts of HIV-1-Puro vector particles progressively abrogated the restriction of N-GFP infection in CV-1 cells. In contrast, no effect on unrestricted B-GFP infection was observed. At saturating levels of the HIV-1-Puro vector, the restriction was completely abrogated such that N-MLV and B-MLV were equivalently infectious. Similarly, HIV-1 virus-like particles (VLPs) that lacked a genome were also capable of complete abrogation of N-MLV restriction. As is shown in Figure 3B, incubation of HIV-1 Gag-Pol VLPs with CV-1 target cells enhanced N-GFP infection such that its titration curve was linear and superimposable on that of B-GFP (compare Figure 3B with Figure 1). Moreover, both HIV-2 particles (Figure 3C) and SIVmac particles (Figure 3D) abrogated N-MLV restriction in CV-1 cells; in both cases, the inclusion of excess lentivirus particles completely restored N-GFP infection to the level obtained with B-GFP. Importantly, these results show that factors responsible for the N-MLV restriction in AGM cells can be saturated by three primate lentiviruses. Figure 3.Abrogation of N-MLV restriction in CV-1 cells by primate lenti virus particles. (A) Abrogation of N-GFP restriction in CV-1 cells by HIV-1 particles. CV-1 cells were inoculated with a fixed and equivalent dose of N-GFP or B-GFP reporter virus (as measured on MDTF cells) in the presence of increasing amounts of abrogating HIV-1-Puro particles. Abrogating virus dose is given as infectious units (i.u.) on TE671 cells. The percentage of N-GFP- (filled symbols) and B-GFP- (open symbols) infected cells is shown. (B) Complete abrogation of N-MLV restriction by HIV-1 particles. N-GFP (filled symbols) and B-GFP (open symbols) were titrated in CV-1 cells, precisely as in Figure 1, except that inoculation was performed in the presence of saturating levels (100 ng of p24) of genome-less HIV-1 VLPs. (C and D) Abrogation of N-MLV restriction by HIV-2 and SIVmac particles. CV-1 cells were inoculated with fixed and equivalent doses of N-GFP (filled symbols) and B-GFP (open symbols), in the presence of increasing amounts of abrogating HIV-2-Puro (C) or SIV-LacZ (D) vector particles. Abrogating virus dose is given as the titer (i.u.) as measured on TE671 cells. Download figure Download PowerPoint Restriction factors in both human and AGM restrict a divergent non-primate lentivirus As the above data indicate, AGM cell lines have the remarkable ability to restrict infection by widely divergent primate and murine retroviruses, perhaps using a common factor, while human cells appear capable of restricting only N-MLV. We next tested a divergent non-primate lentivirus, equine infectious anemia virus (EIAV), for restriction in both human (TE671) and AGM (CV-1) cell lines. As is shown in Figure 4A, titration of an EIAV-GFP vector on TE671 cells gave rise to a curve that was non-linear at high levels of inoculum, a property that is indicative of saturable restriction. To address this more specifically, we asked whether the inclusion of additional EIAV VLPs that lacked a genome could enhance low m.o.i. infection of TE671 cells by the EIAV-GFP vector, i.e. abrogate restriction. As shown in Figure 4B, increasing amounts of EIAV VLPs indeed enhanced EIAV-GFP infection, up to 5-fold. We then showed that EIAV VLPs are also able to saturate Ref1-mediated restriction of N-MLV (Figure 4C). N-GFP infection is restored to the same level as that obtained with B-GFP, In contrast, unrestricted B-GFP infection was unaffected by EIAV VLPs. Figure 4.Restriction of EIAV in human and AGM cells by the same factors that restrict primate lentiviruses and N-MLV. (A) Non-linear titration curve upon infection of human (TE671) cells by EIAV. TE671 cells were inoculated with an increasing dose of EIAV-GFP. The diagonal guide line indicates a slope of 1. (B) Abrogation of EIAV restriction in TE671 cells by EIAV particles. TE671 cells were inoculated with a fixed dose of EIAV-GFP in the presence of increasing levels of EIAV VLPs. (C) Abrogation of N-MLV restriction in TE671 cells by EIAV particles. TE671 cells were inoculated with fixed and equivalent doses of N-GFP or B-GFP in the presence of increasing levels of EIAV VLPs (given in ml of transfected cell supernatant). (D) Non-linear titration curve upon infection of CV-1 cells by EIAV. CV-1 cells were inoculated with an increasing dose of EIAV-GFP, and the percentage of infected cells is plotted. (E and F) Abrogation of EIAV restriction in CV-1 cells by EIAV and HIV-1 particles. CV-1 cells were inoculated with a fixed dose of EIAV-GFP in the presence of increasing levels of (E) EIAV VLPs or (F) HIV-1-Puro particles (given in ng of p24). The percentage of EIAV-GFP infected cells as a function of abrogating particle dose is plotted. (G) Abrogation of N-MLV restriction in CV-1 cells by EIAV particles. CV-1 cells were inoculated with fixed and equivalent doses of N-GFP (filled symbols) or B-GFP (open symbols), as determined by titration on MDTF cells, in the presence of increasing levels of EIAV VLPs. The percentage of MLV-GFP-infected cells is plotted. In the absence of a convenient assay for measuring EIAV VLP concentration, a single stock of EIAV VLP-containing supernatant was used in each of the experiments presented herein, and the VLP level is given as a volume (ml) of this stock. However, each experiment was performed at least twice with independent VLP stocks, with similar results. Download figure Download PowerPoint AGM CV-1 cells also restricted infection by EIAV-GFP. As is shown in Figure 4D, the EIAV-GFP vector titration curve exhibited noticeable non-linearity at high levels of inoculum. In addition, EIAV VLPs significantly enhanced (10-fold) low m.o.i. infection by EIAV-GFP (Figure 4E). We then asked whether saturation of Lv1 with HIV-1 particles could abrogate EIAV restriction in CV-1 cells. As is shown in Figure 4F, this indeed proved to be the case. As with EIAV particles, saturating levels of HIV-1-Puro particles enhanced EIAV infection by 10-fold. In addition, we determined whether EIAV particles could abrogate the N-GFP restriction in CV-1 cells. In fact, EIAV particles were able to abrogate N-GFP restriction completely, and restored the levels of N-GFP infection to the same as those of B-MLV (Figure 4G). Thus, it appears that factor(s) in CV-1 cells are able to restrict this equine retrovirus in addition to primate and murine retroviruses. Primate lentivirus particles do not significantly abrogate Ref1 restriction of N-GFP in human cells As a test of specificity, we examined whether HIV-1 and SIVmac particles could abrogate N-GFP restriction in human cells. Like AGM cells, human cells restrict N-GFP infection but, unlike AGM cells, human cells do not restrict HIV-1, HIV-2 or SIVmac (Figure 2A; Towers et al., 2000; Besnier et al., 2002; Cowan et al., 2002). Therefore, primate lentiviruses are, presumably, not recognized by Ref1 in human cells. Thus, if abrogation of N-MLV restriction by lentiviruses (Figures 3 and 4) is due to saturation of shared restriction factors, then primate lentiviruses should not exhibit this activity in human cells. Indeed, as is shown in Figure 5, N-GFP restriction was not abrogated by either HIV-1-Puro, HIV-2-Puro or SIV-LacZ vector particles in human TE671 cells. Similar results were obtained in a second N-GFP-restricting human cell line, HeLa (data not shown). Figure 5.Primate lentiviruses do not significantly abrogate Ref1-mediated N-MLV restriction in human cells. Human TE671 cells were inoculated with fixed and equivalent doses [in MDTF infectious units (i.u.)] of N-GFP (filled symbols) or B-GFP (open symbols) in the presence of increasing doses of abrogating HIV-1-Puro (A), HIV-2-Puro (B) or SIV-lacZ (C) particles. Higher doses of N-GFP and B-GFP were used in (B) and (C) than in (A). The percentage of MLV-GFP-infected cells is plotted as a function of abrogating virus dose given as TE671 i.u. Download figure Download PowerPoint Partial abrogation of HIV-1 and SIVmac restriction by N-MLV particles If, as the above data suggest, a restricted virus can abrogate the restriction of any other restricted virus, then we reasoned that N-MLV particles should reciprocally abrogate restriction of lentivirus infection. To address this, we inoculated CV-1 cells with a low, restricted dose of HIV-1-GFP or SIV-GFP reporter viruses in the presence of increasing amounts of abrogating N-Neo and B-Neo vector particles. As is shown in Figure 6A, N-Neo particles were indeed able to enhance both HIV-1 and SIVmac infection of CV-1 cells. Again this enhancement was specific, in that B-Neo particles were not able to abrogate restriction. To demonstrate further that this effect was specific to cells that restrict both primate lentiviruses and N-GFP, we simultaneously performed the same experiment using an owl monkey cell line (OMK). This cell line potently restricts HIV-1 infection but does not restrict N-GFP (Figure 1). In this case, HIV-1 infection was not enhanced by N-Neo or B-Neo particles (Figure 6A), but was enhanced >50-fold by HIV-1 VLPs (Figure 6B). Thus, abrogation of HIV-1 restriction by MLV requires that both viruses be sensitive to restriction. These data suggest that the AGM but not the owl monkey form of Lv1 can be saturated by N-MLV. It is of note that the N-Neo-mediated abrogation of HIV-1 and SIVmac restriction in CV-1 cells was incomplete. In fact, N-Neo particles enhanced HIV-1-GFP and SIV-GFP infection of CV-1 cells by only 3- to 6-fold (Figure 6A), while HIV-1 VLPs enhanced infection of each virus by up to 20- to 30-fold (Figure 6B). Increasing the amount of abrogating N-Neo particles in these assays (up to 107 infectious units/well) did not enhance restricted HIV-1-GFP or SIV-GFP infection further beyond that shown in Figure 6A. Figure 6.Abrogation of lentivirus restriction by N-MLV particles. (A) Abrogation of Lv1 restriction in CV-1 but not OMK cells by N-MLV particles. CV-1 and OMK cells were infected with a fixed dose of HIV-1-GFP or SIV-GFP, as indicated, in the presence of increasing doses of abrogating N-Neo (filled symbols) or B-Neo (open symbols) particles. The percentage of HIV-1-GFP- or SIV-GFP-infected cells as a function of abrogating MLV dose is plotted. Abrogating virus dose is given as infectious units (i.u.) as measured on MDTF cells. (B) Abrogation of Lv1 restriction by HIV-1 particles. CV-1 and OMK cells, as indicated, were infected with a fixed dose of HIV-1-GFP or SIV-GFP as in (A) in the presence of the indicated levels (given as ng of p24) of genome-less HIV-1 VLPs. (C) Abrogation of EIAV restriction in human cells by N-MLV particles. TE671 cells were infected with a fixed dose of EIAV-GFP in the presence of increasing amounts of N-Neo (filled symbols) or B-Neo (open symbols) particles. The percentage of EIAV-GFP-infected cells is plotted. Download figure Download PowerPoint We also tested whether MLV particles could abrogate restriction of EIAV infection in human cells. As is shown in Figure 6C, treatment of TE671 cells with Ref1-restricted N-Neo particles enhanced infection by EIAV-GFP ∼5-fold, while treatment with unrestricted B-Neo particles had no effect. These data indicate that saturation of Ref1 in human cells can abrogate restriction of a lentivirus. Evidence for polymorphism in Lv1 among African green monkeys To examine whether the reciprocal abrogation of N-MLV and primate lentiviruses in AGM cells is unique to the CV-1 cell line, we compared the restriction properties of this cell line with two additional cell lines derived from AGMs. Each of these cell lines is derived from the same tissue (kidney) but from a separate AGM subspecies; the BS-C-1 cell line is derived from Cercopithicus aethiops aethiops (grivet), whereas Vero is derived from Cercopithicus aethiops pygerythrus (vervet). The precise origin of CV-1 cells is unclear but, based on characteristic polymorphisms in the CCR5 gene, is most probably derived from Cercopithicus aethiops tantalus (Tantalus) (Kuhmann et al., 2000). Despite their distinct origins, each of these lines potently restricts N-GFP infection (Figure 1), indicating that the presence of the restriction factor is conserved in this species. We examined whether restrictions to HIV-1 and SIVmac infection that are evident in CV-1 cells are conserved in AGM cell lines. HIV-1-GFP and SIV-GFP stocks were normalized to give nearly identical infectivity on non-restricting human TE671 cells (Figure 7A). Thereafter, titration in AGM cells revealed both subtle and more dramatic differences in their relative susceptibility to HIV-1-GFP and SIV-GFP. In particular, both viruses appeared restricted in CV-1 and BS-C-1, with SIV-GFP slightly more so than HIV-1-GFP in CV-1 and less so in BS-C-1 (Figure 7A). In contrast, Vero cells restricted HIV-1-GFP infection to an ∼50-fold greater degree than SIV-GFP, with the latter virus appearing only modestly restricted. As is shown in Figure 7B, the differen" @default.
• W2113203969 created "2016-06-24" @default.
• W2113203969 creator A5066892149 @default.
• W2113203969 date "2003-02-03" @default.
• W2113203969 modified "2023-10-16" @default.
• W2113203969 title "Restriction of multiple divergent retroviruses by Lv1 and Ref1" @default.
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