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ABSTRACT: Despite structural knowledge of broadly neutralizing monoclonal antibodies (NMAbs) complexed to HIV-1 gp120 and gp41 envelope glycoproteins, virus inactivation mechanisms have been difficult to prove, in part because neutralization assays are complex and were previously not understood. Concordant with recent evidence that HIV-1 titers are determined by a race between entry of cell-attached virions and competing inactivation processes, we show that NMAb 2G12, which binds to gp120 N-glycans with α (1, 2)-linked mannose termini and inhibits replication after passive transfer into patients, neutralizes by slowing entry of adsorbed virions. Accordingly, apparent neutralization is attenuated when a kinetically competing virus inactivation pathway is blocked. Moreover, removing 2G12 from media causes its dissociation from virions coupled to accelerated entry and restored infectivity, demonstrating the reversibility of neutralization. A difference between 2G12 dissociation and infectivity recovery rates implies that the inhibited complexes at virus-cell junctions contain several 2G12's that must dissociate before entry commences. Quantitative microscopy of 2G12 binding and dissociation from single virions and studies using a split CCR5 coreceptor suggest that 2G12 competitively inhibits interactions between gp120's V3 loop and the tyrosine sulfate-containing CCR5 amino terminus, thereby reducing assembly of complexes that catalyze entry. These results reveal a unique reversible kinetic mechanism for neutralization by an antibody that binds near a critical V3 region in the glycan shield of gp120.
Proceedings of the National Academy of Sciences 04/2012; 109(20):7829-34. · 9.68 Impact Factor
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ABSTRACT: By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20- to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.
Journal of Virology 03/2010; 84(6):3106-10. · 5.40 Impact Factor
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ABSTRACT: The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.
Journal of Virology 06/2009; 83(16):8289-92. · 5.40 Impact Factor
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ABSTRACT: Vif(IIIB), which has been a standard model for the viral infectivity factor of human immunodeficiency virus type 1 (HIV-1), binds the cytidine deaminase APOBEC3G (A3G) and induces its degradation, thereby precluding its lethal incorporation into assembling virions. Additionally, Vif(IIIB) less efficiently degrades A3F, another potent anti-HIV-1 cytidine deaminase. Although the APOBEC3 paralogs A3A, A3B, and A3C have weaker anti-HIV-1 activities and are only partially degraded by Vif(IIIB), we found that Vif(IIIB) induces their emigration from the nucleus to the cytosol and thereby causes net increases in the cytosolic concentrations and anti-HIV-1 activities of A3A and A3B. In contrast, some other Vifs, exemplified by Vif(HXB2) and Vif(ELI-1), much more efficiently degrade and thereby neutralize all APOBEC3s. Studies focused mainly on A3F imply that it occurs associated with mRNA-PABP1 in translationally active polysomes and to a lesser extent in mRNA processing bodies (P-bodies). A3F appears to stabilize the P-bodies with which it is associated. A correspondingly small proportion of Vif(IIIB) also localizes in P-bodies in an A3F-dependent manner. Stress causes A3A, A3B, A3C, and A3F to colocalize efficiently with Vif(IIIB) and mRNA-PABP1 complexes in stress granules in a manner that is prevented by cycloheximide, an inhibitor of translational elongation. Coimmunoprecipitation studies suggest that Vifs from different HIV-1 isolates associate with all tested APOBEC3s. Thus, Vifs interact closely with structurally diverse APOBEC3s, with effects on their subcellular localization, degradation rates, and antiviral activities. Cytosolic APOBEC3-Vif complexes are predominantly bound to mRNAs that dynamically move between translationally active and storage or processing pools.
Journal of Virology 02/2008; 82(2):987-98. · 5.40 Impact Factor
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ABSTRACT: Binding of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120 to the CCR5 co-receptor reduces constraints on the metastable transmembrane subunit gp41, thereby enabling gp41 refolding, fusion of viral and cellular membranes, and infection. We previously isolated adapted HIV-1(JRCSF) variants that more efficiently use mutant CCR5s, including CCR5(Delta18) lacking the important tyrosine sulfate-containing amino terminus. Effects of mutant CCR5 concentrations on HIV-1 infectivities were highly cooperative, implying that several may be required. However, because wild-type CCR5 efficiently mediates infections at trace concentrations that were difficult to measure accurately, analyses of its cooperativity were not feasible. New HIV-1(JRCSF) variants efficiently use CCR5(HHMH), a chimera containing murine extracellular loop 2. The adapted virus induces large syncytia in cells containing either wild-type or mutant CCR5s and has multiple gp120 mutations that occurred independently in CCR5(Delta18)-adapted virus. Accordingly, these variants interchangeably use CCR5(HHMH) or CCR5(Delta18). Additional analyses strongly support a novel energetic model for allosteric proteins, implying that the adaptive mutations reduce quaternary constraints holding gp41, thus lowering the activation energy barrier for membrane fusion without affecting bonds to specific CCR5 sites. In accordance with this mechanism, highly adapted HIV-1s require only one associated CCR5(HHMH), whereas poorly adapted viruses require several. However, because they are allosteric ensembles, complexes with additional co-receptors fuse more rapidly and efficiently than minimal ones. Similarly, wild-type HIV-1(JRCSF) is highly adapted to wild-type CCR5 and minimally requires one. The adaptive mutations cause resistances to diverse entry inhibitors and cluster appropriately in the gp120 trimer interface overlying gp41. We conclude that membrane fusion complexes are allosteric machines with an ensemble of compositions, and that HIV-1 adapts to entry limitations by gp120 mutations that reduce its allosteric hold on gp41. These results provide an important foundation for understanding the mechanisms that control membrane fusion and HIV-1's facile adaptability.
Journal of Molecular Biology 12/2007; 374(1):64-79. · 4.00 Impact Factor
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ABSTRACT: HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(Delta 18) (Platt et al., J. Virol. 2005, 79: 4357-68).
We have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(Delta 18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(Delta 18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Delta 18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(Delta 18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(Delta 18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion.
We propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 - an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide.
Retrovirology 02/2007; 4:55. · 6.47 Impact Factor
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ABSTRACT: Abstract
Background
HIV-1 envelope glycoprotein (Env) induces membrane fusion as a result of sequential binding to CD4 and chemokine receptors (CCR5 or CXCR4). The critical determinants of CCR5 coreceptor function are the N-terminal domain (Nt) and the second extracellular loop. However, mutations in gp120 adapt HIV-1 to grow on cells expressing the N-terminally truncated CCR5(Δ18) (Platt et al ., J. Virol . 2005, 79: 4357–68).
Results
We have functionally characterized the adapted Env (designated Env(NYP)) using a quantitative cell-cell fusion assay. The rate of fusion with target cells expressing wild-type CCR5 and the resistance to fusion inhibitors was virtually identical for wild-type Env and Env(NYP), implying that the coreceptor affinity had not increased as a result of adaptation. In contrast, Env(NYP)-induced fusion with cells expressing CCR5(Δ18) occurred at a slower rate and was extremely sensitive to the CCR5 binding inhibitor, Sch-C. Resistance to Sch-C drastically increased after pre-incubation of Env(NYP)- and CCR5(Δ18)-expressing cells at a temperature that was not permissive to fusion. This indicates that ternary Env(NYP)-CD4-CCR5(Δ18) complexes accumulate at sub-threshold temperature and that low-affinity interactions with the truncated coreceptor are sufficient for triggering conformational changes in the gp41 of Env(NYP) but not in wild-type Env. We also demonstrated that the ability of CCR5(Δ18) to support fusion and infection mediated by wild-type Env can be partially reconstituted in the presence of a synthetic sulfated peptide corresponding to the CCR5 Nt. Pre-incubation of wild-type Env- and CCR5(Δ18)-expressing cells with the sulfated peptide at sub-threshold temperature markedly increased the efficiency of fusion.
Conclusion
We propose that, upon binding the Nt region of CCR5, wild-type Env acquires the ability to productively engage the extracellular loop(s) of CCR5 – an event that triggers gp41 refolding and membrane merger. The adaptive mutations in Env(NYP) enable it to more readily release its hold on gp41, even when it interacts weakly with a severely damaged coreceptor in the absence of the sulfopeptide.
Retrovirology. 01/2007;
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ABSTRACT: Deoxycytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family) have RNA-binding motifs, invade assembling human immunodeficiency virus (HIV-1), and hypermutate reverse transcripts. Antagonistically, HIV-1 viral infectivity factor degrades these enzymes. A3G is enzymatically inhibited by binding RNA within an unidentified large cytosolic ribonucleoprotein, implying that RNA degradation during reverse transcription may activate intravirion A3G at the necessary moment. We purified a biologically active tandem affinity-tagged A3G from human HEK293T cells. Mass spectrometry and coimmunoprecipitation from HEK293T and T lymphocyte extracts identified many RNA-binding proteins specifically associated with A3G and A3F, including poly(A)-binding proteins (PABPs), YB-1, Ro-La, RNA helicases, ribosomal proteins, and Staufen1. Most strikingly, nearly all A3G-associated proteins were known to bind exclusively or intermittently to translating and/or dormant mRNAs. Accordingly, A3G in HEK293T and T lymphocyte extracts was almost completely in A3G-mRNA-PABP complexes that shifted reversibly between polysomes and dormant pools in response to translational inhibitors. For example arsenite, which inhibits 5'-cap-dependent translational initiation, shifted mRNA-A3G-PABP from polysomes into stress granules in a manner that was blocked and reversed by the elongation inhibitor cycloheximide. Immunofluorescence microscopy showed A3G-mRNA-PABP stress granules only partially overlapping with Staufen1. A3G coimmunoprecipitated HIV-1 RNA and many mRNAs. Ribonuclease released nearly all A3G-associated proteins, including A3G homo-oligomers and A3G-A3F hetero-oligomers, but the viral infectivity factor remained bound. Many proteins and RNAs associated with A3G are excluded from A3G-containing virions, implying that A3G competitively partitions into virions based on affinity for HIV-1 RNA.
Journal of Biological Chemistry 10/2006; 281(39):29105-19. · 4.77 Impact Factor
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Hana Golding,
Surender Khurana,
Felix Yarovinsky,
Lisa R King,
Galina Abdoulaeva,
Liselotte Antonsson,
Christer Owman,
Emily J Platt, David Kabat,
John F Andersen,
Alan Sher
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ABSTRACT: Molecular mimicry of chemokine ligands has been described for several pathogens. Toxoplasma gondii produces a protein, cyclophilin-18 (C-18), which binds to the human immunodeficiency virus (HIV) co-receptor CCR5 and inhibits fusion and infection of T cells and macrophages by R5 viruses but not by X4 viruses. We recently identified structural determinants of C-18 required for anti-HIV activity (Yarovinsky, F., Andersen, J. F., King, L. R., Caspar, P., Aliberti, J., Golding, H., and Sher, A. (2004) J. Biol. Chem. 279, 53635-53642). Here we have elucidated the fine specificity of CCR5 residues involved in binding and HIV inhibitory potential of C-18. To delineate the regions of CCR5 involved in C-18 binding, we analyzed C-18 inhibition of cells expressing CXCR4/CCR5 chimeric receptors and CCR5 with a truncated N terminus (Delta2-19). These experiments identified a critical role for the N terminus of CCR5 in C-18 binding and anti-HIV activity. Studies with a large panel of CCR5 N-terminal peptides, including Tyr-sulfated analogues, truncated peptides, and alanine-scanning mutants, suggested that each of the 12-17 amino acids in the N terminus of CCR5 are essential for C-18 binding and inhibitory activity. Tyr sulfation did not improve C-18 reactivity. This finding is of interest because the same CCR5 N-terminal region was shown previously to play a key role in binding of HIV-1 envelope glycoproteins. The elucidation of the functional C-18-binding mechanism may help in the rational design of novel antiviral agents against HIV.
Journal of Biological Chemistry 09/2005; 280(33):29570-7. · 4.77 Impact Factor
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ABSTRACT: APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.
AIDS Research and Human Retroviruses 08/2005; 21(7):611-9. · 2.25 Impact Factor
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ABSTRACT: Replication of human immunodeficiency virus type 1 (HIV-1) in diverse conditions limiting for viral entry into cells frequently leads to adaptive mutations in the V3 loop of the gp120 envelope glycoprotein. This has suggested that the V3 loop limits the efficiencies of HIV-1 infections, possibly by directly affecting gp120-coreceptor affinities. In contrast, V3 loop mutations that enable HIV-1(JR-CSF) to use the low-affinity mutant coreceptor CCR5(Y14N) are shown here to have negligible effects on the virus-coreceptor affinity but to dramatically accelerate the irreversible conformational conversion of the envelope gp41 subunits from a three-stranded coil into a six-helix bundle. This slow step is blocked irreversibly by the inhibitor T-20. To further evaluate the role of entry rates in controlling infection efficiencies and viral adaptations, we developed methods to quantitatively measure viral entry kinetics. The virions were adsorbed by spinoculation at 4 degrees C onto HeLa-CD4/CCR5 cell clones that either had limiting or saturating concentrations of CCR5. After warming to 37 degrees C, the completion of entry was monitored over time by the resistance of infections to the competitive CCR5 inhibitor TAK-779. Our results suggest that the efficiency of entry of cell-attached infectious HIV-1 is principally controlled by three kinetic processes. The first is a lag phase that is caused in part by the concentration-dependent reversible association of virus with CD4 and CCR5 to form an equilibrium assemblage of complexes. Second, this assembly step lowers but does not eliminate a large activation energy barrier for a rate-limiting, CCR5-dependent conformational change in gp41 that is sensitive to blockage by T-20. The rate of infection therefore depends on the fraction of infectious virions that are sufficiently saturated with CCR5 to undergo this conformational change and on the magnitude of the activation energy barrier. Although only a small fraction of fully assembled viral complexes overcome this barrier per hour, the ensuing steps of entry are rapidly completed within 5 to 10 min. Thus, this barrier limits the overall flow rate at which the attached virions enter cells, but it has no effect on the lag time that precedes this entry flow. Third, a relatively rapid and kinetically dominant process of viral inactivation, which may partly involve endocytosis, competes with infectious viral entry. Our results suggest that the V3 loop of gp120 has a major effect on the rate-limiting coreceptor-dependent conformational change in gp41 and that adaptive viral mutations, including V3 loop mutations, function kinetically by accelerating this inherently slow step in the entry pathway.
Journal of Virology 05/2005; 79(7):4347-56. · 5.40 Impact Factor
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ABSTRACT: By selecting the R5 human immunodeficiency virus type 1 (HIV-1) strain JR-CSF for efficient use of a CCR5 coreceptor with a badly damaged amino terminus [i.e., CCR5(Y14N)], we previously isolated variants that weakly utilize CCR5(Delta18), a low-affinity mutant lacking the normal tyrosine sulfate-containing amino-terminal region of the coreceptor. These previously isolated HIV-1(JR-CSF) variants contained adaptive mutations situated exclusively in the V3 loop of their gp120 envelope glycoproteins. We now have weaned the virus from all dependency on the CCR5 amino terminus by performing additional selections with HeLa-CD4 cells that express only a low concentration of CCR5(Delta18). The adapted variants had additional mutations in their V3 loops, as well as one in the V2 stem (S193N) and four alternative mutations in the V4 loop that eliminated the same N-linked oligosaccharide from position N403. Assays using pseudotyped viruses suggested that these new gp120 mutations all made strong contributions to use of CCR5(Delta18) by accelerating a rate-limiting CCR5-dependent conformational change in gp41 rather than by increasing viral affinity for this damaged coreceptor. Consistent with this interpretation, loss of the V4 N-glycan at position N403 also enhanced HIV-1 use of a different low-affinity CCR5 coreceptor with a mutation in extracellular loop 2 (ECL2) [i.e., CCR5(G163R)], whereas the double mutant CCR5(Delta18,G163R) was inactive. We conclude that loss of the N-glycan at position N403 helps to convert the HIV-1 envelope into a hair-trigger form that no longer requires strong interactions with both the CCR5 amino terminus and ECL2 but efficiently uses either site alone. These results demonstrate a novel functional role for a gp120 N-linked oligosaccharide and a high degree of adaptability in coreceptor usage by HIV-1.
Journal of Virology 05/2005; 79(7):4357-68. · 5.40 Impact Factor
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ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to infect macrophages. Previous experiments have mapped viral determinants of macrophage infectivity to the V3 hypervariable region of the HIV-1 envelope glycoprotein. In our earlier studies, V1 and V2 sequences of HIV-1 were also shown to alter the ability of virus to spread in macrophage cultures, whereas no effect was seen in lymphocyte cultures. In the present study, determinants that allowed certain HIV-1 clones to infect and spread in macrophages were primarily mapped to the V2 region and were found to act by influencing early events of viral infection. By an assay of viral entry into macrophages, it was shown that viruses with the V2 region from the Ba-L strain of HIV-1 had >10-fold-higher entry efficiency than viruses with the V2 region derived from the NL4-3 strain. V1 region differences between these groups caused a twofold difference in entry. The known low expression of CD4 on macrophages appeared to be important in this process. In entry assays conducted with HeLa cell lines expressing various levels of CD4 and CCR5, low levels of CD4 influenced the efficiency of entry and fusion which were dependent on viral V1 and V2 envelope sequences. In contrast, no effect of V1 or V2 was seen in HeLa cells expressing high levels of CD4. Thus, the limited expression of CD4 on macrophages or other cell types could serve as a selective factor for V1 and V2 envelope sequences, and this selection could in turn influence many aspects of AIDS pathogenesis in vivo.
Journal of Virology 04/2005; 79(8):4828-37. · 5.40 Impact Factor
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Retrovirology. 01/2005;
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ABSTRACT: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) is an antiretroviral deoxycytidine deaminase that lethally hypermutates human immunodeficiency virus type 1 (HIV-1) but is itself neutralized by the HIV-1-encoded viral infectivity factor. Accordingly, APOBEC3G occurs specifically in human T lymphocytic cell lines that contain this antiviral defense, including H9. Since the substrate specificities of related cytidine deaminases are strongly influenced by their intracellular quantities, we analyzed the factors that control APOBEC3G expression. The levels of APOBEC3G mRNA and protein were unaffected by treatment of proliferating H9 cells with interferons or tumor necrosis factor-alpha but were enhanced up to 20-fold by phorbol myristate acetate. This induction was mediated at the transcriptional level by a pathway that required activation of the protein kinase Calpha/betaI isozyme (PKC), mitogen-activated protein kinase kinase (MEK) 1 and 2, and extracellular signal-regulated kinase (ERK). Correspondingly, induction of APOBEC3G was blocked by multiple inhibitors that act at diverse steps of this pathway. The PKCalpha/betaI/MEK/ERK pathway also controlled basal levels of APOBEC3G mRNA and protein, which consequently declined when cells were treated with these inhibitors or arrested in the G(0) state of the cell cycle by serum starvation. We conclude that expression of the antiviral APOBEC3G editing enzyme is dynamically controlled by the PKCalpha/betaI/MEK/ERK protein kinase cascade in human T lymphocytes.
Journal of Biological Chemistry 11/2004; 279(40):41744-9. · 4.77 Impact Factor
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ABSTRACT: A PHQ motif near the amino termini of gammaretroviral envelope glycoprotein surface (SU) subunits is important for infectivity but not for incorporation into virions or binding to cognate receptors. The H residue of this motif is most critical, with all substitutions we tested being inactive. Interestingly, porcine endogenous retroviruses (PERVs) of all three host-range groups, A, B, and C, lack full PHQ motifs, but most members have an H residue at position 10. H10A PERV mutants are noninfectious but were efficiently transactivated by adding to the assays a PHQ-containing SU or receptor-binding subdomain (RBD) derived from a gibbon ape leukemia virus (GALV). A requirement of this transactivation was a functional GALV receptor on the cells. In contrast to this heterologous transactivation, PERV RBDs and SUs were inactive in all tested cells, including porcine ST-IOWA cells. Surprisingly, transactivation by GALV RBD enabled wild-type or H10A mutant PERVs of all three host-range groups to efficiently infect cells from humans and rodents that lack functional PERV receptors and it substantially enhanced infectivities of wild-type PERVs, even for cells with PERV receptors. Thus, PERVs can suboptimally infect cells that contain cognate receptors or they can employ a transactivation pathway to more efficiently infect all cells. This ability to infect cells lacking cognate receptors was previously demonstrated only for nontransmissible variant gammaretroviruses with recombinant and mutant envelope glycoproteins. We conclude that some endogenously inherited mammalian retroviruses also have a receptor-independent means for overcoming host-range and interference barriers, implying a need for caution in xenotransplantation, especially of porcine tissues.
Journal of Virology 09/2004; 78(16):8868-77. · 5.40 Impact Factor
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ABSTRACT: The viral infectivity factor (Vif) of HIV type-1 (HIV-1) is essential for efficient viral replication, yet was, until recently, enigmatic. This resulted from the complexity and cellular specificity of its function and the correspondingly complex systems that are required for its investigation. These limitations have been overcome and Vif function has been rapidly elucidated, with implications for the development of drugs to block its activity. These studies have revealed a novel component of the innate immune system, APOBEC3G, that lethally hypermutates retroviruses, including HIV-1. For HIV-1, the competition between the virus and APOBEC3G is tipped in favor of the invader by Vif, which binds to APOBEC3G and triggers its polyubiquitination and rapid degradation, thereby preventing its entry into progeny virions.
Trends in Molecular Medicine 07/2004; 10(6):291-7. · 10.35 Impact Factor
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ABSTRACT: The viral infectivity factor (Vif) encoded by HIV-1 neutralizes a potent antiviral pathway that occurs in human T lymphocytes and several leukemic T-cell lines termed nonpermissive, but not in other cells termed permissive. In the absence of Vif, this antiviral pathway efficiently inactivates HIV-1. It was recently reported that APOBEC3G (also known as CEM-15), a cytidine deaminase nucleic acid-editing enzyme, confers this antiviral phenotype on permissive cells. Here we describe evidence that Vif binds APOBEC3G and induces its rapid degradation, thus eliminating it from cells and preventing its incorporation into HIV-1 virions. Studies of Vif mutants imply that it contains two domains, one that binds APOBEC3G and another with a conserved SLQ(Y/F)LA motif that mediates APOBEC3G degradation by a proteasome-dependent pathway. These results provide promising approaches for drug discovery.
Nature Medicine 12/2003; 9(11):1398-403. · 22.46 Impact Factor
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ABSTRACT: A widely dispersed interference group of retroviruses that includes the feline endogenous virus (RD114), baboon endogenous virus (BaEV), human endogenous virus type W (HERV-W), and type D primate retroviruses uses the human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) as a common cell surface receptor. Although hamster cells are fully resistant to these viruses and murine cells are susceptible only to BaEV and HERV-W pseudotype viruses, these rodent cells both become highly susceptible to all of the viruses after treatment with tunicamycin, an inhibitor of protein N-linked glycosylation. A partial explanation for these results was recently provided by findings that the orthologous murine transporter mASCT2 is inactive as a viral receptor, that a related (ca. 55% identity) murine paralog (mASCT1; gene name, SLC1A4) mediates infections specifically of BaEV and HERV-W, and that N-deglycosylation of mASCT1 activates it as a receptor for all viruses of this interference group. Because the only two N-linked oligosaccharides in mASCT1 occur in the carboxyl-terminal region of extracellular loop 2 (ECL2), it was inferred that this region contributes in an inhibitory manner to infections by RD114 and type D primate viruses. To directly and more thoroughly investigate the receptor active sites, we constructed and analyzed a series of hASCT2/mASCT2 chimeras and site-directed mutants. Our results suggest that a hypervariable sequence of 21 amino acids in the carboxyl-terminal portion of ECL2 plays a critical role in determining the receptor properties of ASCT2 proteins for all viruses in this interference group. In addition, we analyzed the tunicamycin-dependent viral susceptibility of hamster cells. In contrast to mASCT1, which contains two N-linked oligosaccharides that partially restrict viral infections, hamster ASCT1 contains an additional N-linked oligosaccharide clustered close to the others in the carboxyl-terminal region of ECL2. Removal of this N-linked oligosaccharide by mutagenesis enabled hamster ASCT1 to function as a receptor for all viruses of this interference group. These results strongly suggest that combinations of amino acid sequence changes and N-linked oligosaccharides in a critical carboxyl-terminal region of ECL2 control retroviral utilization of both the ASCT1 and ASCT2 receptors.
Journal of Virology 04/2003; 77(5):2936-45. · 5.40 Impact Factor
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ABSTRACT: The viral infectivity factor (Vif) of human immunodeficiency virus type 1 (HIV-1) neutralizes an unidentified antiviral pathway that occurs only in nonpermissive (NP) cells. Using a yeast two-hybrid screen of a human lymphocyte cDNA library, we identified several potential Vif partners. One, the nuclear body protein Sp140, was found specifically in all NP cells (n = 12 cell lines tested; P < or = 0.001), and HIV-1 infection induced its partial dispersal from nuclear bodies into cytosolic colocalization with Vif. Our results implicate Sp140 in a response to HIV-1 that may be related to or coordinated with the pathway that inactivates HIV-1 lacking vif.
Journal of Virology 11/2002; 76(21):11133-8. · 5.40 Impact Factor
