Klaus Strebel, Ph.D.Building 4, Room 3124 Memorial DriveBethesda, MD 20892-0460Phone: 301-496-3132Fax: firstname.lastname@example.org
Klaus Strebel received his Ph.D. in microbiology in 1985 from the University of Heidelberg, Germany. He joined the NIAID Laboratory of Molecular Microbiology (LMM) in 1986 as a postdoctoral fellow and is now head of the Viral Biochemistry Section within LMM.
Primate immunodeficiency viruses, including HIV-1, are characterized by the presence of a number of viral accessory genes that encompass vif, vpr, vpx, vpu, and nef. The vif, vpr, and nef genes are expressed in most HIV-1, HIV-2, and SIV isolates. In contrast, the vpu gene is found exclusively in HIV-1 and one SIV isolate, SIVcpz. The vpx gene, on the other hand, is not found in HIV-1 isolates but is common to HIV-2 and most SIV isolates. Defects in accessory genes are frequently not correlated with a detectable impairment of virus replication in continuous cell lines, in contrast to primary cell types, which more closely reflect the in vivo situation. The molecular basis for this cell-type-specific role of some of the accessory proteins remains largely unknown. However, it becomes increasingly clear that these proteins indeed exert important functions in their relevant target cells in vivo and, in fact, most if not all of the HIV accessory proteins exert multiple independent functions. The precise biochemical activity for most of the accessory proteins is currently not well understood; however, there is increasing evidence to suggest that they do not have catalytic activity on their own but rather function as adaptor molecules that connect other viral or cellular factors to various cellular pathways.
The primary objective of the section is to investigate the biological and biochemical functions of HIV accessory proteins, in particular Vif and Vpu, and to define their role in virus replication. This includes both the detailed biological and biochemical characterization of the individual viral proteins as well as the investigation of their engagement with distinct host factors. Understanding the molecular mechanisms of viral proteins will benefit our general understanding of the HIV replication process and may reveal new targets for antiviral therapy. In addition, the investigation of virus-host interactions will give us new insights into general cellular processes involved in protein trafficking, degradation, post-translational modification, and so forth.
The HIV-Accessory Protein Vpu
Parallel with the discovery of vpu as a functional gene in 1987, we realized that its gene product, an 81-residue integral membrane protein, had an important function in regulating virus release (Strebel, 1989). These early studies were soon complemented by the realization that Vpu had a second, independent function during virus replication that resulted in the rapid degradation of CD4 receptor molecules from the endoplasmic reticulum in HIV-infected cells (Willey, 1992). One of the main goals of our subsequent research was to understand the molecular mechanisms involved in Vpu-mediated virus release and CD4 degradation. We found that CD4 degradation required a specific interaction of Vpu with newly synthesized CD4 molecules in the endoplasmic reticulum (Bour, 1995). This interaction was dependent on sequences in the cytoplasmic domains of CD4 and Vpu. The interaction of Vpu and CD4 was found to trigger a process that resulted in the rapid degradation of CD4. Using a yeast-two-hybrid-based screening of human cDNA libraries, we identified, in collaboration with Richard Benarous and his group at the Institute Cochin, France, a novel Vpu-binding protein, human-bTrCP (h-bTrCP), which functions as a ubiquitin ligase and connects CD4 to the proteolytic machinery (Margottin, 1998). Two essential structural motifs were identified in h-bTrCP: a C-terminal domain composed of seven WD repeats that is important for protein-protein interactions and an F-box motif located at the N-terminus that is functional in mediating interactions with Skp1, a protein recently identified as a targeting factor for ubiquitin-mediated proteolysis. We were able to show both in vivo and in vitro that the WD repeats mediate binding to Vpu (Margottin 1998). Interestingly, we found that h-bTrCP interacts with Vpu only when the latter is phosphorylated at serine 52 and 56, providing the first explanation as to why these highly conserved residues in Vpu are of crucial importance for CD4 degradation. The F-box of h-bTrCP, on the other hand, was found to mediate interactions with Skp1.
Figure 1: Vpu-induced degradation of CD4 involves TrCP (view enlarged illustration)
Vpu can bind to newly synthesized CD4 in the endoplasmic reticulum. In addition, Vpu has a binding domain for TrCP. This domain includes two conserved serine residues in the cytoplasmic domain of Vpu that are constitutively phosphorylated by casein kinase 2 (CK-2). Formation of multiprotein complexes consisting of TrCP, Vpu, and CD4 results in the TrCP-dependent ubiquitination of CD4 and subsequent degradation by cytosolic proteasomes.
Based on our observation that Vpu inhibits TrCP-dependent degradation of IkB and thus blocked NF-kB activation (Bour, 2001), we explored the possible involvement of Vpu in HIV-1-induced apoptosis (Akari, 2001). We found that in HIV-1-infected CD4+ T cell lines as well as primary CD4+ T cells, expression of Vpu significantly contributed to the induction of apoptosis. Using an inducible expression system we found that the effect of Vpu on apoptosis was direct and did not require the co-expression of other viral proteins. Analysis of cellular factors involved in the induction of apoptosis demonstrated that Vpu down-modulated the NF-kB-dependent expression of anti-apoptotic genes such as Bcl-xL and A1/Bfl-1. Concomitantly, Vpu expression resulted in increased levels of active caspase-3. These effects of Vpu involved an interaction with TrCP as evidenced by the fact that mutation of the TrCP binding motif in Vpu abolished its apoptogenic potential. These results suggest that Vpu promotes apoptosis through its inhibition of NF-kB.
Figure 2: Mechanism of Vpu-induced apoptosis. (view enlarged image)
In unstimulated cells, NF-kB resides in the cytoplasm in an inactive complex with its inhibitor IkB. Upon stimulation of cells by cytokines such as TNF-a (1), IkB is rapidly phosphorylated by an IkB specific kinase (2), which results in the rapid degradation of IkB via a TrCP-dependent pathway (3). Infection of cells by HIV-1 results in the gradual intracellular accumulation of Vpu. Because of its constitutively active TrCP-binding motif and the fact that it is not sensitive to TrCP-mediated proteolysis, Vpu functions as a competitive inhibitor of TrCP. This results in the gradual accumulation of IkB and the progressive impairment of the cell’s ability to activate NF-kB (4). The inhibition of NF-kB blocks the synthesis of anti-apoptotic proteins such as the Bcl-2 family proteins (e.g., Bcl-xL and A1/Bfl-1) or TNFR complex proteins (e.g., TRAF1) (5). TRAF1 is induced by TNF-a treatment and normally inhibits activation of caspase-8 (6). In Vpu-expressing cells, the levels of TRAF1, in response to TNF stimulation, are reduced and no longer sufficient to inhibit the cytokine-induced activation of caspase-8 (6). Activated caspase-8 in turn induces the release of cytochrome c from the mitochondria (7). Release of cytochrome c is normally inhibited by the Bcl-2 family of proteins. However, in Vpu-expressing cells the levels of Bcl-2 proteins are limiting and no longer sufficient to block cytochrome c release (8). After its release from the mitochondria, cytochrome c forms ternary complexes with Apaf-1 and caspase-9 (9), resulting in the activation of caspase-3 (10). Active caspase-3 finally triggers a reaction that results in the cleavage of a number of target proteins including Bcl-2 family proteins (11) and leads to cell death (12).
The HIV-Accessory Protein Vif
The HIV-1 Vif protein plays an important role in regulating virus infectivity (18). The lack of a functional Vif protein results in the production of virions with reduced or abolished infectivity (Strebel, 1987). This effect of Vif on virus infectivity is producer cell dependent and can vary by several orders of magnitude. Virus replication in non-permissive cell types such as H9 is strictly dependent on Vif, while Vif-defective viruses can efficiently replicate in permissive hosts such as Jurkat cells. The cellular factors determining the requirement for Vif are currently not known. Results from heterokaryon analyses, which involved the fusion of restrictive with permissive cell types suggested the presence of an inhibitory factor in restrictive cell types. However, the identity of the proposed inhibitory factor and its mode of action remain elusive. Recent work investigating the ability of Vif from different lentiviruses for cross-species trans-complementation suggested that Vif itself functions in a host-cell dependent manner, supporting the notion that Vif may interact with as yet unknown cellular factors.
Although vif genes are present in all lentiviruses, with the exception of equine infectious anemia virus, there is relatively little sequence conservation between different Vif variants. Nevertheless, HIV-1 Vif was found to be capable of functionally complementing Vif-defective HIV-2 and SIVmac isolates, suggesting common functional domains and a common mode of action. Similarly, HIV-2 Vif was capable of complementing HIV-1 Vif defects.
HIV-1 Vif associates with the intermediate filament vimentin
A significant amount of Vif can be found in association with the intermediate filament network in virus-producing cells (Karczewski, 1996); however, the domain(s) in Vif responsible for this association as well as its functional significance remain to be determined.
Figure 3: Colocalization of Vif with the intermediate filament vimentin.
HeLa cells were transfected with the Vif expression vector pNL-A1. To better visualize colocalization of Vif with vimentin, cells were treated with demecolcine (0.1 mg/ml) for 15 hours following transfection. Demecolcine treatment of HeLa cells results in a structural reorganization of vimentin intermediate filaments to form a nuclear cage structure. Cells were stained for vimentin (green) and Vif (red) Colocalization of Vif and vimentin is apparent by the yellow color. A Nomarski bright field image is shown in the right panel.
HIV-1 Vif is packaged into the nucleoprotein complex through an interaction with viral genomic RNA
Despite the severe impact of Vif defects on virus infectivity, its mechanism of action has thus far remained obscure. It is generally accepted that Vif-deficient viruses can attach to and penetrate host cells but are blocked at a post-penetration step early in the infection cycle. Yet, comparison of virion morphology or protein composition between wild type and Vif-defective virions has thus far been inconclusive and produced conflicting results. Several reports have suggested that Vif affects the stability of the viral nucleoprotein complex. In particular, NC and reverse transcriptase were found to be less stably associated with viral cores in the absence of Vif. Nevertheless, Vif is generally believed to function within the virus-producing cell. This assumption was largely based on the observation that relatively small amounts of Vif were found to be packaged, with estimates ranging from less than 1 to 100 molecules of Vif per virion. Furthermore, packaging of Vif into virus particles was generally believed to be nonspecific, leading to questions as to the functional significance of Vif incorporation into virions.
Based on this information, we performed an in-depth biochemical analysis of Vif in purified virions from permissive or restrictive host cells to investigate the specificity of Vif incorporation into virions (Khan, 2001). Pulse/chase analysis of single-cycle infected H9 cells did not reveal any Vif-dependent differences in viral protein processing and maturation consistent with recent reports by other investigators. Instead, detergent extraction of purified virions demonstrated an association of Vif with the nucleoprotein complex (Khan, 2001). Quantitative analyses suggest that approximately 10 to 15 percent of total Vif protein is packaged into virions (Khan, 2001). This efficiency is comparable to the 5 percent packaging efficiency reported for the HIV-1 Env protein (Willey, 1988). Interestingly, HIV-1 variants carrying mutations in the nucleocapsid zinc finger domains abolished Vif packaging. In addition, an RNA-packaging defective virus was significantly impaired in packaging of Vif. Finally, deletion of a putative RNA-binding motif between residues 75-114 in Vif abolished its packaging into virions. Virion-associated Vif was resistant to detergent extraction and co-purified with components of the viral nucleoprotein complex and functional reverse transcription complexes (Khan, 2001). Thus, Vif is specifically packaged into virions as a component of the viral nucleoprotein complex. Our data suggest that the specific association of Vif with the viral nucleoprotein complex might be functionally significant and could be a critical requirement for infectivity of viruses produced from restrictive host cells.
Intravirion processing of Vif
In investigating the specificity of Vif packaging into virions derived from various cell lines, we noticed that Vif is subjected to intra-virion processing by the HIV-1 protease (Pr) (Khan, 2002). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type-independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral protease and was sensitive to protease inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala150. Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and simian immunodeficiency virus Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also had no effect on Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function suggesting that intravirion processing of Vif is important for the production of infectious viruses.
HIV-1 Vif is efficiently packaged into virions during productive but not chronic infection
Packaging of the HIV type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. To investigate this apparent discrepancy, we compared the packaging efficiency of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion) while packaging into virions from chronically infected H9 cells was near the limit of detection (4 to 6 copies of Vif per virion) (Kao, 2003). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild type virus was readily packaged (30to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild type infectivity (Kao, 2003). These results provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.
Ritu Goila, Ph.D., Visiting Fellow, 301-496-3132, email@example.com; Sandra Kao, M.S., Biologist, 301-496-3132, firstname.lastname@example.org; Mohammod Khan, Ph.D., Research Fellow, 301-496-3132, email@example.com; Eri Miyagi, Ph.D., Visiting Fellow, 301-496-3132, firstname.lastname@example.org; Sandrine Opi, Ph.D., Visiting Fellow, 301-496-3132, email@example.com; Hiroaki Takeuchi, Ph.D., Visiting Fellow, 301-496-3132, firstname.lastname@example.org.
(View list in PubMed.)
Strebel K. HIV-1 Vpu; Putting a Channel to the TASK. Mol Cell. 2004. 14: 150.
Xu H, Svarovskaia ES, Barr R, Zhang Y, Khan MA, Strebel K, Pathak VK. A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion. Proc Natl Acad Sci USA. 2004. 101: 5652.
Nguyen KL, llano M, Akari H, Miyagi E, Poeschla EM, Strebel K, Bour S. Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression. Virology. 2004. 319: 163.
Akari H, Fujita M, Kao S, Khan MA, Shehu-Xhilaga M, Adachi A, Strebel K. 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. J Biol Chem. 2004. 279: 12355.
Markovic I, Stantchev TS, Fields KH, Tiffany LJ, Tomic M, Weiss CD, Broder CC, Strebel K, Clouse KA. Thiol/disulfide exchange is a prerequisite for CXCR4-tropic HIV-1 envelope-mediated T-cell fusion during viral entry. Blood. 2004. 103: 1586.
Strebel K. Virus-host interactions: role of HIV proteins Vif, Tat, and Rev. AIDS. 2003. 17 Suppl 4: S25-34.
Varthakavi V, Smith RM, Bour SP, Strebel K, Spearman P. Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production. Proc Natl Acad Sci USA. 2003. 100: 15154.
Kao S, Khan MA, Miyagi E, Plishka R, Buckler-White A, Strebel K. The human immunodeficiency virus type 1 Vif protein reduces intracellular expression and inhibits packaging of APOBEC3G (CEM15), a cellular inhibitor of virus infectivity. J Virol. 2003. 77: 11398.
Bour S, Strebel K. The HIV-1 Vpu protein: a multifunctional enhancer of viral particle release. Microbes Infect. 2003. 5: 1029.
Bour S, Akari H, Miyagi E, Strebel K. Naturally occurring amino acid substitutions in the HIV-2 ROD envelope glycoprotein regulate its ability to augment viral particle release. Virology. 2003. 309: 85.
Kao S, Akari H, Khan MA, Dettenhofer M, Yu XF, Strebel K. Human immunodeficiency virus type 1 Vif is efficiently packaged into virions during productive but not chronic infection. J Virol. 2003. 77: 1131.
Khan MA, Akari H, Kao S, Aberham C, Davis D, Buckler-White A, Strebel K. Intravirion processing of the human immunodeficiency virus type 1 Vif protein by the viral protease may be correlated with Vif function. J Virol. 2002. 76: 9112.
Ma C, Marassi FM, Jones DH, Straus SK, Bour S, Strebel K, Schubert U, Oblatt-Montal M, Montal M, Opella SJ. Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1. Protein Sci. 2002. 11: 546.
Akari H, Bour S, Kao S, Adachi A, Strebel K. The human immunodeficiency virus type 1 accessory protein Vpu induces apoptosis by suppressing the nuclear factor kappaB-dependent expression of antiapoptotic factors. J Exp Med. 2001. 194: 1299.
Khan MA, Aberham C, Kao S, Akari H, Gorelick R, Bour S, Strebel K. Human immunodeficiency virus type 1 Vif protein is packaged into the nucleoprotein complex through an interaction with viral genomic RNA. J Virol. 2001. 75: 7252.
Bour S, Perrin C, Akari H, Strebel K. The human immunodeficiency virus type 1 Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated degradation of Ikappa B. J Biol Chem. 2001. 276: 15920.
Akari H, Arold S, Fukumori T, Okazaki T, Strebel K, Adachi A. Nef-induced major histocompatibility complex class I down-regulation is functionally dissociated from its virion incorporation, enhancement of viral infectivity, and CD4 down-regulation. J Virol. 2000. 74: 2907.
Bour S, Strebel K. HIV accessory proteins: multifunctional components of a complex system. Adv Pharmacol. 2000. 48: 75.
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Last Updated August 20, 2012