Lentiviral Vif: Viral Hijacker of the Ubiquitin-Proteasome System

Since the beginning of time, microorganisms have been devising ways to bypass detection and destruction by our immune system. Therefore, it is no surprise that along with the identification of the cellular antiviral protein APOBEC3G (A3G) has come the recognition of the viral solution to this assault. Here, we review the research that led up to the identification of A3G and the mechanism that the human immunodeficiency virus protein Vif developed to evade A3G's antiviral activities.     

重组腺病毒表达载体介导艾滋病毒Vif蛋白调控KSHV潜伏感染的初探

目的:利用AdEasy腺病毒载体系统包装含人类免疫缺陷病毒1型(艾滋病毒:HIV-1)病毒体感染性因子(Vif)的重组腺病毒,使之感染原发性渗出性淋巴瘤细胞系BCBL-1细胞,并检测Vif蛋白对细胞中卡波济肉瘤相关疱疹病毒(Kaposi'ssarcomaassociated herpesvirus,KSHV)潜伏感染与...     

Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding domain-stabilized hydrophobic interface in Vif

APOBEC3G (A3G) and related cytidine deaminases are potent inhibitors of retroviruses. HIV-1 Vif hijacks the cellular Cul5-E3 ubiquitin ligase to degrade APOBEC3 proteins and render them ineffective against these viruses. Here, we report that HIV-1 Vif is a novel zinc-binding protein containing an H-x(5)-C-x(17-18)-C-x(3-5)-H motif that is distinct from other recognized classes of zinc fingers. Zinc-binding stabilized a conserved hydrophobic interface within the HCCH motif that is critical for Vif-Cul5 E3 assembly and Vif function. An N-terminal region in the first Cullin repeat of Cul5, which is dispensable for adaptor ElonginC binding, was required for interaction with Vif. This region is the most divergent sequence between Cul2 and Cul5, a factor that may contribute to the selection of Cul5 and not Cul2 by Vif. This is the first example of a zinc-binding substrate receptor responsible for the assembly of a Cullin-RING ligase, representing a new target for antiviral development.     

Production of infectious virus and degradation of APOBEC3G are separable functional properties of human immunodeficiency virus type 1 Vif

HIV-1 Vif regulates viral infectivity by inhibiting the encapsidation of APOBEC3G (APO3G) through proteasomal degradation of the protein. Here we compared various Vif proteins for their ability to induce APO3G degradation and rescue viral infectivity. We found that Vif expressed from proviral vectors caused relatively inefficient degradation of APO3G in HeLa cells yet was very effective in inhibiting APO3G's antiviral activity. On the other hand, Vif expressed autonomously from a codon-optimized vector caused very efficient APO3G degradation and also effectively inhibited APO3G's antiviral effects. In contrast, a Vif chimera containing an N-terminal fluorescent tag efficiently induced APO3G degradation but was unable to restore viral infectivity. The lack of a direct correlation between APO3G degradation and rescue of viral infectivity suggests that these two properties of Vif are functionally separable. Our data imply that intracellular degradation of APO3G may not be the sole activity of Vif required for the production of infectious virions from APO3G-expressing cells.     

HIV-1 Vif promotes the formation of high molecular mass APOBEC3G complexes

HIV-1 Vif inhibits the antiviral activity of APOBEC3G (APO3G) by inducing proteasomal degradation. Here, we studied the effects of Vif on APO3G in vitro. In this system, Vif did not cause APO3G degradation. Instead, Vif induced changes in APO3G that affected immunoprecipitation of the native protein. This effect required wt Vif and was reversed by heat denaturation of APO3G. Sucrose gradient analysis demonstrated that wt Vif induced the gradual transition of APO3G translated in vitro or expressed in HeLa cells from a low molecular mass conformation to puromycin-sensitive high molecular mass (HMM) complexes. In the absence of Vif or the presence of biologically inactive Vif APO3G failed to form HMM complexes. Our results expose a novel function of Vif that promotes the assembly of APO3G into presumably packaging-incompetent HMM complexes and may explain how Vif can overcome the APO3G-imposed block to HIV replication under conditions of no or inefficient APO3G degradation.     

HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.     

Novel targets for HIV therapy

There are currently 25 drugs belonging to 6 different inhibitor classes approved for the treatment of human immunodeficiency virus (HIV) infection. However, new anti-HIV agents are still needed to confront the emergence of drug resistance and various adverse effects associated with long-term use of antiretroviral therapy. The 21st International Conference on Antiviral Research, held in April 2008 in Montreal, Canada, therefore featured a special session focused on novel targets for HIV therapy. The session included presentations by world-renowned experts in HIV virology and covered a diverse array of potential targets for the development of new classes of HIV therapies. This review contains concise summaries of discussed topics that included Vif-APOBEC3G, LEDGF/p75, TRIM 5α, virus assembly and maturation, and Vpu. The described viral and host factors represent some of the most noted examples of recent scientific breakthroughs that are opening unexplored avenues to novel anti-HIV target discovery and validation, and should feed the antiretroviral drug development pipeline in the near future.     

Human APOBEC3G incorporation into murine leukemia virus particles

The human APOBEC3G protein exhibits broad antiretroviral activity against a variety of retroviruses. It is packaged into viral particles and executes its antiviral function in the target cell. The packaging of APOBEC3G into different viral particles requires a mechanism that confers this promiscuity. Here, APOBEC3G incorporation into murine leukemia virus (MLV) was studied using retroviral vectors. APOBEC3G uptake did not require either its cytidine deaminase activity or the presence of a retroviral vector genome. Results from immunoprecipitation and co-localization studies of APOBEC3G with a MLV Gag-CFP (cyan fluorescent protein) fusion protein imply an interaction between both proteins. RNase A treatment did not inhibit the co-precipitation of Gag-CFP and APOBEC3G, suggesting that the interaction is RNA independent. Like human immunodeficiency virus (HIV) Gag, the MLV Gag precursor protein appears to interact with APOBEC3G, indicating that Gag contains conserved structures which are used to encapsidate APOBEC3G into different retroviral particles.     

Changes in the Vif protein of HIV-1 associated with the development of resistance to inhibitors of viral protease

The protease (PR) and virus infectivity factor (vif) gene sequences of a cohort of HIV-1 infected patients showing evidence of developing protease inhibitor (PI) resistance whilst undergoing highly active antiretroviral therapy (HAART) have been determined. The PR sequences showed the presence of the classical mutations associated with resistance to PIs. The sequence of the Vif protein showed less variation in samples from PI treated patients than in specimens prepared from treatment-naive patients. In addition a number of amino acid positions within Vif showed highly significant preferences for a particular amino acid in the PI-treated cohort compared to the untreated control cohort.     

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).