Random Mutagenesis MAPPIT Analysis Identifies Binding Sites for Vif and Gag in Both Cytidine Deaminase Domains of Apobec3G

The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultanously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.     

High level expression of human immunodeficiency virus type-1 Vif inhibits viral infectivity by modulating proteolytic processing of the Gag precursor at the p2/nucleocapsid processing site

The human immunodeficiency virus type-1 Vif protein has a crucial role in regulating viral infectivity. However, we found that newly synthesized Vif is rapidly degraded by cellular proteases. We tested the dose dependence of Vif in non-permissive H9 cells and found that Vif, when expressed at low levels, increased virus infectivity in a dose-dependent manner. Surprisingly, however, the range of Vif required for optimal virus infectivity was narrow, and further increases in Vif severely reduced viral infectivity. Inhibition of viral infectivity at higher levels of Vif was cell type-independent and was associated with an accumulation of Gag-processing intermediates. Vif did not act as a general protease inhibitor but selectively inhibited Gag processing at the capsid and nucleocapsid (NC) boundary. Identification of Vif variants that were efficiently packaged but were unable to modulate Gag processing suggests that Vif packaging was necessary but insufficient for the production of 33- and 34-kDa processing intermediates. Interestingly, these processing intermediates, like Vif, associated with viral nucleoprotein complexes more rigidly than mature capsid and NC. We conclude that virus-associated Vif inhibits processing of a subset of Gag precursor molecules at the p2/NC primary cleavage site. Modulation of processing of a small subset of Gag molecules by physiological levels of Vif may be important for virus maturation. However, the accumulation of such processing intermediates at high levels of Vif is inhibitory. Thus, rapid intracellular degradation of Vif may have evolved as a mechanism to prevent such inhibitory effects of Vif.     

Insulin-like growth factor II mRNA binding protein 1 associates with Gag protein of human immunodeficiency virus type 1, and its overexpression affects virus assembly

The assembly of human immunodeficiency virus type 1 (HIV-1) particles is driven by viral Gag protein. This function of Gag not only benefits from its self-multimerization property but also depends on its interaction with a number of cellular factors such as TSG101 and ALIX/AIP1 that promote virus budding and release from cell surfaces. However, interaction with Gag also allows some cellular factors such as APOBEC3G and Trim5alpha to access viral replication machinery and block viral replication. In this study, we report a new HIV-1 Gag-binding factor named insulin-like growth factor II mRNA binding protein 1 (IMP1). Gag-IMP1 interaction requires the second zinc finger of the nucleocapsid (NC) domain of Gag and the KH3 and KH4 domains of IMP1. A fourfold reduction of HIV-1 infectivity was seen with overexpression of the wild-type IMP1 and its mutant that is able to interact with Gag but not with overexpression of IMP1 mutants exhibiting Gag-binding deficiency. The decreased viral infectivity was further shown as a result of diminished viral RNA packaging, abrogated Gag processing on the cellular membranes, and impeded maturation of virus particles. Together, these results demonstrate that IMP1 interacts with HIV-1 Gag protein and is able to block the formation of infectious HIV-1 particles.     

The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.     

The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures.

The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.     

Human immunodeficiency virus type 1 Vif protein binds to the Pr55Gag precursor.

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes. Previous reports suggest that vif-deleted viruses are limited in replication because of a defect in the late steps of the virus life cycle. One of the remaining questions is to determine whether the functional role of Vif involves a specific interaction with virus core proteins. In this study, we demonstrate a direct interaction between Vif and the Pr55Gag precursor in vitro as well as in infected cells. No interaction is observed between Vif and the mature capsid protein. The Pr55Gag-Vif interaction is detected (i) in the glutathione S-transferase system, with in vitro-translated proteins demonstrating a critical role of the NC p7 domain of the Gag precursor; (ii) with proteins expressed in infected cells; and (iii) by coimmunoprecipitation experiments. Deletion of the C-terminal 22 amino acids of Vif abolishes its interaction with the Pr55Gag precursor. Furthermore, point mutations in the C-terminal domain of Vif which have been previously shown to abolish virus infectivity and binding to cell membranes dramatically decrease the Gag-Vif interaction. These results suggest that the interaction between Vif and the pr55Gag precursor is a critical determinant of Vif function.     

Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis

The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G–Apobec3G and the Apobec3G–Vif interactions, the mammalian protein–protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G–Apobec3G interaction. All mutations that inhibit Apobec3G–Apobec3G binding also inhibit the Apobec3G–Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G–Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer.     

Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions.     

Human immunodeficiency virus type 1 Vif does not influence expression or virion incorporation of gag-, pol-, and env-encoded proteins.

The Vif protein of human immunodeficiency virus type 1 is required for productive replication in peripheral blood lymphocytes and a limited number of immortalized T-lymphoid lines (nonpermissive cells). In contrast, Vif is fully dispensable for virus replication in other T-cell lines (permissive cells). Because the infection phenotype of released virions is determined by producer cells and by the presence of Vif in those cells, we have analyzed the protein contents of purified viral particles in an attempt to define compositional differences that could explain the infection phenotype. Surprisingly, we were unable to discern any Vif- or cell-type-dependent quantitative or qualitative difference in the Gag, Pol, and Env proteins of virions or virus-producing cells that correlates with virus infectivity. We were, however, able to demonstrate that Vif itself is present in virions and, using semiquantitative Western blotting (immunoblotting), that there is an average of 30 to 80 molecules of Vif incorporated into each virion. Importantly, parallel analyses of total lysates of the producer cells revealed that the cell-associated expression levels of Vif are close to those of the Gag proteins. Given the dramatically higher abundance of Vif in cells than in virions, we speculate that Vif exerts its principal activity during the processes of virus assembly and budding and that this function could be of a structural-conformational nature.     

Equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release

A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.     

Vpr.A3A chimera inhibits HIV replication

Several APOBEC3 proteins (A3F and A3G), that are cytidine deaminases restrict human immunodeficiency virus (HIV) replication in the absence of the viral infectivity factor (Vif) protein. However, Vif leads to their degradation and counteracts their effects. Another member, A3A, restricts some retrotransposons and another virus but not HIV. We reasoned that this failure was due to the lack of appropriate targeting. Thus, we fused A3A to another viral protein, Vpr, which binds p6 in Gag and is incorporated into viral cores. Indeed, the Vpr.A3A chimera but not A3A was found abundantly in the viral core. It also restricted potently the replication of HIV and simian immunodeficiency virus in the presence and absence of Vif. Because we identified a high frequency of G to A mutations in viral cDNAs, this antiviral activity was mediated by DNA editing. Interestingly, our fusion protein did not restrict murine leukemia virus, which does not incorporate Vpr. Thus, by targeting appropriately a potent single domain cytidine deaminase, we rendered HIV and simian immunodeficiency virus restriction resistant to Vif.     

TSG101在病毒出芽过程中的作用及其机制

TSG101基因是新发现的抑癌基因候选者,定位于人类 11号染色体 p1511-p1512,其编码产物TSG101蛋白N端区域与泛素结合酶(UBC)同源。近年来研究发现,TSG101基因具有多种重要的功能,与多种病毒出芽密切相关,所以TSG101可作为一个新的抗病毒靶点。本文主要从TSG101在多种病毒（HIV、IAV、MARV、ASV等）出芽过程中扮演的角色,TSG101与多种蛋白(泛素、Nedd4、ARMMs、Tom1、Gag、VP40、NP等)的相互作用进而辅助病毒出芽的机制,以及TSG101抑制剂的研究等方面进行阐述。     

The viral infectivity factor (Vif) of HIV-1 unveiled

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.     

Regulation of Apobec3F and human immunodeficiency virus type 1 Vif by Vif-Cul5-ElonB/C E3 ubiquitin ligase

The human cytidine deaminase Apobec3F (h-A3F), a protein related to the previously recognized antiviral factor Apobec3G (h-A3G), has antiviral activity against human immunodeficiency virus type 1 (HIV-1) that is suppressed by the viral protein Vif. The mechanism of HIV-1 Vif-mediated suppression of h-A3F is not fully understood. Here, we demonstrate that while h-A3F, like h-A3G, was able to suppress primate lentiviruses other than HIV-1 (simian immunodeficiency virus from African green monkeys [SIVagm] and Rhesus macaques [SIVmac]), the interaction between Vif proteins and h-A3F appeared to differ from that with h-A3G. H-A3F showed no change in its species specificity against HIV-1 or SIVagm Vif when a negatively charged amino acid was replaced with a lysine at position 128, a residue critical for h-A3G recognition by HIV-1 Vif. However, HIV-1 Vif, but not SIVagm Vif, was able to bind h-A3F and induce its polyubiquitination and degradation through the Cul5-containing E3 ubiquitin ligase. Interference with Cul5-E3 ligase function by depletion of Cul5, through RNA interference or overexpression of Cul5 mutants, blocked the ability of HIV-1 Vif to suppress h-A3F. A BC-box mutant of HIV-1 Vif that failed to recruit Cul5-E3 ligase but was still able to interact with h-A3F failed to suppress h-A3F. Interestingly, interference with Cul5-E3 ligase function or overexpression of h-A3F or h-A3G also increased the stability of HIV-1 Vif, suggesting that like the substrate molecules h-A3F and h-A3G, the substrate receptor protein Vif is itself also regulated by Cul5-E3 ligase. Our results indicate that Cul5-E3 ligase appears to be a common pathway hijacked by HIV-1 Vif to defeat both h-A3F and h-A3G. Developing inhibitors to disrupt the interaction between Vif and Cul5-E3 ligase could be therapeutically useful, allowing multiple host antiviral factors to suppress HIV-1.     

The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.     

Murine retrovirus escapes from murine APOBEC3 via two distinct novel mechanisms

APOBEC3G (A3G) is an antiretroviral host factor that functions by deaminating dC to dU in retroviral cDNA. HIV-1 Vif protein counteracts A3G via a ubiquitin-proteasome pathway. In the case of a simple retrovirus such as the murine leukemia virus (MLV), it remains unclear why it can replicate in cells expressing APOBEC3 (A3) even though it doesn't possess any accessory proteins such as Vif. In this study, we demonstrate that MLV escapes from murine A3 (mA3) via two distinct novel mechanisms. First, viral RNA (vRNA) blocks the binding of mA3 to Gag, resulting in the exclusion of mA3 from MLV virions. Second, viral protease (vPR) cleaves mA3 after maturation of virions. Here, we suggest that each virus has its own strategy to escape from A3 proteins and that these mechanisms might be used by other viruses that do not possess Vif-like protein. On the other hand, mice possess another form of mA3, delta exon5, that escapes from the cleavage by vPR to show more antiviral activity than the wild type mA3. This also suggests that battles between host intrinsic immunity and viruses have led to the evolution of proteins on both sides.