Primate immunodeficiency viruses, including HIV-1, are characterized by the presence of accessory genes such as vif, vpr, vpx, vpu, and nef. Current knowledge indicates that none of these proteins has enzymatic activity. Instead, they interact with cellular ligands either to act as adapter molecules to redirect the normal function of host factors for virus-specific purposes or to inhibit a normal host function by mediating degradation or causing intracellular mislocalization/sequestration of the factors involved.
The primary objective of the Viral Biochemistry Section is to investigate the biological and biochemical functions of HIV accessory proteins, in particular Vif, Vpu, and Vpx, 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.
Our early study on Vpu-induced degradation of the HIV receptor CD4 presented the first example in the HIV literature of the engagement of the host proteasome machinery in the control of cellular proteins by HIV accessory proteins (Willey et al., JVI 66:7193 ). Since then, similar strategies, although mechanistically distinct, were identified for the control of BST-2 by Vpu, APOBEC3G by Vif, or SAMHD1 by the Vpx protein. Thus, the control of host mechanisms through proteolytic degradation of cellular targets is a common theme in HIV biology and remains an intense focus of our research. Modulating the intracellular localization of host factors is another popular strategy employed by viruses to control their function and/or to subvert it to benefit the virus. Examples under investigation in the lab are the down-modulation of BST-2 from the cell surface with subsequent degradation through the lysosomal pathway or the sequestration of APOBEC3G away from the site of virus assembly.
Dr. Strebel received his Ph.D. in microbiology in 1985 from the University of Heidelberg, Germany. After postdoctoral research in Germany on foot-and-mouth disease protein processing and maturation, he joined the Laboratory of Molecular Microbiology (LMM) in 1986 as a postdoctoral fellow to work on molecular mechanisms of HIV-1 replication. He was awarded tenure in 1998 and, since 2000, has been chief of the Viral Biochemistry Section within LMM.
Sandra Kao, M.S., Biologist Eri Miyagi, Ph.D., Staff ScientistJanet Chen, Research FellowKlaus Strebel, Ph.D., Viral Biochemistry Section ChiefHaruka Yoshii, Ph.D., Visiting FellowSarah Welbourn, Ph.D., Visiting Fellow
Miyagi E, Kao S, Yedavalli V, Strebel K. CBFβ enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G. J Virol. 2014 May;88(9):4839-52.
Klase Z, Yedavalli VS, Houzet L, Perkins M, Maldarelli F, Brenchley J, Strebel K, Liu P, Jeang KT. Activation of HIV-1 from latent infection via synergy of RUNX1 inhibitor Ro5-3335 and SAHA. PLoS Pathog. 2014 Mar 20;10(3):e1003997.
Strebel K. HIV accessory proteins versus host restriction factors. Curr Opin Virol. 2013 Dec;3(6):692-9.
Andrew A, Strebel K. HIV-1 accessory proteins: Vpu and Vif. Methods Mol Biol. 2014;1087:135-58.
Welbourn S, Dutta SM, Semmes OJ, Strebel K. Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1. J Virol. 2013 Nov;87(21):11516-24.
Yoshida T, Koyanagi Y, Strebel K. Functional antagonism of rhesus macaque and chimpanzee BST-2 by HIV-1 Vpu is mediated by cytoplasmic domain interactions. J Virol. 2013 Dec;87(24):13825-36.
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Fig. 1: Vpu reduces surface expression of BST-2 by blocking resupply of newly synthesized BST-2.Credit: NIAID
The goal of this project was to define the mechanism by which Vpu antagonizes the antiviral effects of BST-2. We performed a series of kinetic studies in various cell types to determine the fate of newly synthesized or pre-existing BST-2 over time. Our results indicate that surface down-regulation of BST-2 is not due to accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell (Fig. 1). We also confirmed that Vpu can reduce the stability of newly synthesized BST-2 in transfected 293T cells via an endoplasmic reticulum (ER)-associated degradation pathway. However, this effect was dependent on high level (over)expression of Vpu. Surprisingly, virus-encoded Vpu neither affected the stability of newly synthesized BST-2 in transiently transfected 293T cells nor altered the stability of newly synthesized endogenous BST-2 in virus-infected HeLa or CEMx174 cells. Instead, virus-expressed Vpu acted at a post-ER level to increase the turnover of mature BST-2. Our results indicate that Vpu interferes with the trafficking of BST-2 to the cell surface from a post-ER compartment presumably by rerouting BST-2 to the lysosomal compartment for degradation.
Fig. 2: Interactions of Vpu with non-human BST-2 involves cytoplasmic domain interactions.Credit: NIAID
The goal of this study was to characterize the regions in BST-2 that define sensitivity to Vpu. One approach was to transfer sequences from human BST-2 into the Vpu-resistant rhesus BST-2 to check function and Vpu sensitivity of the resulting chimera. Interestingly, mutation of a single residue in the TM domain of rhesus BST-2 (I48T) was sufficient to increase sensitivity to Vpu significantly. This suggests that sensitivity of BST-2 to Vpu can be regulated by relatively subtle changes in the BST-2 TM domain. In a parallel study, we characterized residues in the TM domain of human BST-2 critical for its interaction with HIV-1 Vpu. We found that three amino acid residues (I34, L37, and L41) were critical for the interaction with Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that positioning of these four residues (i.e., I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for Vpu-mediated antagonism of human tetherin. An additional aspect of our research addressed the species-specific function of Vpu. The widely studied NL4-3 Vpu functionally inactivates human BST-2 but not monkey BST-2, leading to the notion that Vpu antagonism is species specific. Interestingly, Vpus encoded by the X4-tropic SHIVDH12 and the R5-tropic SHIVAD8 were capable of enhancing virion release from infected rhesus PBMC to varying degrees. Transfer of the SHIVDH12 Vpu transmembrane domain to the HIV 1NL4-3 Vpu conferred antagonizing activity against macaque BST-2. Inactivation of the SHIVDH12 and SHIVAD8 vpu genes impaired virus replication in six of eight inoculated rhesus macaques, resulting in lower plasma viral RNA loads, slower losses of CD4+ T cells, and delayed disease progression. The expanded host range of the SHIVDH12 Vpu was not due to adaptation during passage in macaques but was an intrinsic property of the parental HIV-1DH12 Vpu protein. These results demonstrate that some HIV-1 isolates encode a Vpu with a broader host-range. Of note, while Vpu interacts with human BST-2 primarily through their respective transmembrane domains, antagonism of rhesus BST-2 by Vpu involved an interaction of their cytoplasmic domains (Fig. 2). Bimolecular fluorescence complementation analysis to detect Vpu-BST-2 interactions suggested that the physical interaction of Vpu with rhesus or chimpanzee BST-2 involves a five-residue motif in the cytoplasmic domain of BST-2 previously identified as important for antagonism of monkey and great ape BST-2 by SIV Nef. Thus, our study identifies a novel mechanism of antagonism of rhesus and great ape BST-2 by Vpu that targets the same motif in BST-2 used by SIV Nef and might explain the expanded host-range observed for Vpu isolates in our previous study.
Fig. 3: BST-2 carrying a C-terminal HA tag localizes in the plasma membrane, as shown in model (c), which is inconsistent with GPI anchor modification.Credit: NIAID
Human BST-2 shares only 33 percent amino acid identity with the rat protein. However, both proteins are predicted by bioinformatics tools to be GPI anchor modified. Given that GPI anchor modification of transmembrane proteins appears to be extremely rare in nature and given the lack of direct experimental evidence for GPI anchor modification of either rat or human BST-2, the goal of our study was to assess GPI anchor modification of human BST-2 experimentally. We used a variety of biochemical assays including PI-PLC treatment, aerolysin treatment, and gradual truncation of the putative GPI anchor signal. In addition, we employed indirect immunofluorescence to detect the positioning of epitope tags on permeabilized or unpermeabilized cells (Fig. 3). We were unable to verify GPI anchor modification of human BST-2. Instead, we found strong evidence that the C-terminal putative GPI anchor signal in human BST-2 represents, in fact, a second TM domain. For instance, the C-terminal putative GPI anchor domain can be transferred to a heterologous protein and function as a TM domain. Also, C-terminally epitope-tagged BST-2 is functional, and the tag is properly located on the cytoplasmic side of the plasma membrane (Fig. 3). Finally, replacing the GPI anchor domain in human BST-2 with the TM domain of heterologous proteins can yield BST-2 capable of inhibiting virus release. To characterize the importance of the BST-2 ectodomain for virus restriction, we added or substituted heterologous coiled-coil or non-coiled-coil sequences in the BST-2 ectodomain. We found that extending or substituting the BST-2 ectodomain using non-coiled-coil sequences inhibited BST-2 function. In contrast, substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Even addition of a coiled-coil to the existing BST-2 structure that almost doubled the size of the BST-2 ectodomain was tolerated. These results suggest that there is significant tolerance with respect to the length of the ectodomain. Overall, we conclude that the size of the BST-2 ectodomain can be reduced or enlarged with heterologous coiled-coil sequences, revealing a significant flexibility in the overall size of the protein while changes to the heptad motifs or the register of the coil had variable effects depending on the positioning of the deletion.
Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remained unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells, while others saw no significant impact of CBFβ on Vif stability. In our own studies, we confirmed that CBFβ increases Vif steady-state levels. Interestingly, CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression post-transcriptionally. Kinetic studies revealed effects of CBFβ both on metabolic stability and on the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We conclude that CBFβ acts like a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G (Fig. 4).
Fig. 4: Model of CBFβ-mediated enhancement of Vif biosynthesis, stabilization of Vif protein, and enhancement of A3G degradation. (A) During Vif biosynthesis, CBFβ binds to nascent Vif polypeptide chains and inhibits their premature termination and degradation as defective ribosomal products (DRiPs) (step 1). This results in increased levels of newly synthesized Vif. CBFβ bound to Vif further stabilizes Vif and reduces intrinsic degradation of Vif by cellular proteasomes (step 2). The result is increased half-life of Vif. (B) Vif binding to A3G is CBFβ-independent. However, only in the presence of CBFβ can Vif effectively assemble a Cul5-based E3 ubiquitin ligase complex (step3) required for ubiquitination and subsequent proteolytic degradation of A3G. Other components of the Cul5 E3 ubiquitin ligase complex were omitted for simplicity.Credit: NIAID
Sterile alpha motif and HD domain protein 1 (SAMHD1) is a recently identified host factor targeted by the HIV-2 and SIVsm encoded Vpx proteins to allow replication of these viruses in myeloid cells. Interestingly, while HIV-1 does not possess a Vpx protein, Vpx also enhances infection of myeloid and dendritic cells as well as resting CD4+ T cells by this virus. In susceptible cell types, SAMHD1 has been shown to restrict infection of these lentiviruses at the reverse transcription step and Vpx counteracts this restriction by binding to and causing the proteasomal degradation of SAMHD1. Mutations in SAMHD1 have been associated with Aicardi-Goutieres Syndrome (AGS), a condition associated with increased production of interferon alpha. While SAMHD1 has recently been shown to possess nucleic acid binding properties and was also reported to have exonuclease activity, its main catalytic activity described to date is a dGTP-dependent dNTPase activity. Thus, SAMHD1 is thought to restrict HIV-1 infection by decreasing the levels of cellular dNTP pools to below that required for reverse transcription.
One interesting observation is that SAMHD1 restriction does not strictly correlate with its expression. Indeed, fully HIV-1-permissive cells, such as activated CD4+ T cells or undifferentiated THP-1 cells, also express high amounts of the SAMHD1 protein. The inability of SAMHD1 to inhibit HIV infection when overexpressed in HeLa or 293T cells combined with the lack of correlation of SAMHD1 expression with restriction activity in myeloid cells raised the question of whether SAMHD1 activity was dependent on a second host factor with a cell-type-specific expression profile or whether SAMHD1 activity was regulated at a post-translational level. We designed two studies to address these questions. First, we cloned SAMHD1 from THP-1 cells to study its potential regulation at the transcriptional or post-transcriptional level. Second, we studied post-translational modifications of SAMHD1 and their role in regulating its biological activity. Thus, the overall goal of this study is to gain mechanistic insights into the regulation of SAMHD1 function.
We identified and characterized two SAMHD1 splice variants that are expressed naturally together with full-length SAMHD1 in a variety of cell types. The splice variants identified either lack exons 8-9 (Δ8-9), eliminating a C-terminal portion of the HD domain, or exon 14 (Δ14). Unlike wild-type SAMHD1, which is stable in the absence of Vpx, both splice variants were inherently unstable and were rapidly degraded even in the absence of Vpx. Neither splice variant exhibited dNTPase activity in an in vitro assay, suggesting they also lack antiviral activity. Nevertheless, the identification of SAMHD1 splice variants exposes a potential regulatory mechanism that could enable a cell to control its dNTP levels by regulating SAMHD1 expression at a post-transcriptional level.
Fig. 5: Effect of SAMHD1 phosphorylation on restriction activity against HIV-1. U937 cells were transduced with pCDH lentiviral particles encoding either WT SAMHD1, the indicated SAMHD1 mutants, or an empty vector. After puromycin selection, cells were differentiated overnight with PMA. Differentiated cells were infected with increasing volumes of VSV-G pseudotyped HIV-1-GFP and the percent infection (% GFP-positive cells) determined by flow cytometry 48 h later. We found that mutation of the major phosphorylation site (T592) to alanine (A) did not affect the antiviral activity of SAMHD1. In contrast, mutation of T592 to glutamate (E), which can act as a phosphomimetic, eliminated antiviral activity. This suggests that phosphorylation of SAMHD1 at T592 inhibits its antiviral activity.Credit: NIAID
Our studies on post-translational modifications of SAMHD1 revealed that it can be phosphorylated at several sites. We found that phosphorylation of SAMHD1 at any of four identified positions did not significantly affect protein stability, localization, or sensitivity to Vpx-mediated degradation. Mutation of any of the phosphorylation sites also had no detectable effect on dNTPase activity of SAMHD1 in vitro. Using orthophosphate labeling and mass spectrometric analyses, we identified T592 as a major site of SAMHD1 phosphorylation. Mutating residue T592 to alanine (T592A) had no effect on SAMHD1 restriction activity. In contrast, replacing residue T592 by the phosphomimetic glutamic acid (T592E) abolished SAMHD1 restriction activity (Fig. 5). Mutation of any of the other phosphorylation sites identified did not modulate the ability of SAMHD1 to restrict HIV-1. Thus, phosphorylation at T592 appears to act as a negative regulator of SAMHD1 restriction activity. Consistent with this, phosphorylation of endogenous SAMHD1 in THP-1 cells was reduced under conditions where the protein can restrict HIV-1 replication, further implicating SAMHD1 phosphorylation as a mechanism to regulate anti-HIV SAMHD1 function.
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Last Updated July 15, 2014