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Xiao Fang Yu - One of the best experts on this subject based on the ideXlab platform.
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determinants of lentiviral Vpx crl4 e3 ligase mediated samhd1 degradation in the substrate adaptor protein dcaf1
Biochemical and Biophysical Research Communications, 2019Co-Authors: Nannan Zhang, Xiao Fang Yu, Siyu ShenAbstract:Abstract The lentiviral accessory protein Vpx enhances viral replication in macrophages, dendritic cells and resting CD4+ T cells by utilizing the host CRL4-DCAF1 E3 ligase to trigger the degradation of the intrinsic antiviral factor SAMHD1. Distinct from the species-specific recognition of either the N or C-terminus of SAMHD1 by Vpx proteins of different HIV-2 and SIV lineages, Vpx recruits SAMHD1 onto the same CRL4-DCAF1 complex. However, the determinants in DCAF1 that are required for Vpx-mediated SAMHD1 degradation have not been well characterized. Here, we demonstrate that the viral protein Vpx is resistant to suppression by a cellular inhibitor of the CRL4-DCAF1 E3 ligase, Merlin/NF2, through targeting a separate binding region in DCAF1. The Merlin binding-deficient DCAF1 truncation mutant (1–1417) is sufficient for Vpx-CRL4-DCAF1 E3 ligase assembly and SAMHD1 degradation. We found that the carboxyl-terminus ED-rich region (1312–1417) of DCAF1 is required for the nuclear localization of DCAF1 and for the Vpx-DCAF1 interaction. We identified the DCAF1 (1–1311) truncation mutant as a dominant negative mutant of wild-type DCAF1 that inhibits Vpx-mediated SAMHD1 degradation. These results suggest a unique strategy by which Vpx exploits DCAF1 to counteract this host restriction factor.
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inhibition of Vpx mediated samhd1 and vpr mediated host helicase transcription factor degradation by selective disruption of viral crl4 dcaf1 e3 ubiquitin ligase assembly
Journal of Virology, 2017Co-Authors: Hong Wang, Richard B Markham, Jiaming Su, Wenwen Zheng, Wenyan Zhang, Zhaolong Li, Xiao Fang YuAbstract:ABSTRACT The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or host helicase transcription factor (HLTF), respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that posttranslational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelator N , N , N ′-tetrakis-(2′-pyridylmethyl)ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac (simian immunodeficiency virus of macaques) infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HLTF degradation or Vpr-induced G 2 cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel anti-human immunodeficiency virus (HIV) drug development. IMPORTANCE The Vpr and its paralog Vpx are accessory proteins encoded by different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx- or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with its substrate. Selective disruption of Vpx- or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration using N , N , N ′-tetrakis-(2′-pyridylmethyl)ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-dependent cellular protein functions. Therefore, information obtained from this study may be important for novel anti-HIV drug development.
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Research Progress in Viral Protein Vpx induction of Proteasomal Degradation of the Antiviral Factor SAMHD1
Chinese journal of virology, 2016Co-Authors: Juan Du, Ke Zhao, Xiao Fang YuAbstract:HIV the pathogen responsible for the transmission of AIDS, which is associated with a high mortality rate. Vpx is expressed in HIV-2/SIV and promotes retroviral infection in specific cells. This promotion is achieved by Vpx-induced formation of the CRL4E3 complex, which removes the endogenous SAMHD1 via proteasomal degradation. Multiple domains of SAMHD1(e.g., N-terminus, nuclear localization signal, linker, HD domain, and C-terminus)are essential for Vpx-induced degradation.HIV-1that does not express Vpx is also evolved with mechanisms to bypass or suppress the antiviral function of SAMHD1,such as the tolerance against a low level of dNTPs and induction of SAMHD1 degradation through cyclin L2.Based on previous reports published chronologically, as well as the latest findings in the field, this review focuses on the mechanism of Vpx-mediated degradation of SAMHD1,and its promotion of HIV-1infection.
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HD domain of SAMHD1 influences Vpx-induced degradation at a post-interaction step.
Biochemical and Biophysical Research Communications, 2016Co-Authors: Jian Kang, Ke Zhao, Xiao Fang Yu, Juan DuAbstract:Primate SAMHD1 proteins are potent inhibitors of viruses, including retroviruses such as HIV-1, HIV-2, and SIV. Vpx, a distinctive viral protein expressed by HIV-2 and some SIVs, induces SAMHD1 degradation by forming a Vpx-DCAF1-based ubiquitin ligase complex. Either the N- or the C-terminus of SAMHD1 is critical for Vpx-induced degradation, depending on the types of SAMHD1 and Vpx proteins. However, it was not fully understood whether other regions of SAMHD1 also contribute to its depletion by Vpx. In the present study, we report that SAMHD1 from chicken (SAMHD1GG) was not degraded by SIVmac Vpx, in contrast with results for human SAMHD1 (SAMHD1HS). Results regarding to SAMHD1HS and SAMHD1GG fusion proteins supported previous findings that the C-terminus of SAMHD1HS is essential for Vpx-induced degradation. Internal domain substitution, however, revealed that the HD domain also contributes to Vpx-mediated SAMHD1 degradation. Interestingly, the HD domain influenced Vpx-mediated SAMHD1 degradation without affecting Vpx-SAMHD1 interaction. Therefore, our findings revealed that factors in addition to Vpx-SAMHD1 binding influence the efficiency of Vpx-mediated SAMHD1 degradation.
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variation of two primate lineage specific residues in human samhd1 confers resistance to n terminus targeted siv Vpx proteins
Journal of Virology, 2014Co-Authors: Richard B Markham, Xiao Fang YuAbstract:Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection in myeloid cells but is inactivated by certain classes of simian immunodeficiency virus (SIV) Vpx proteins. Vpx proteins recruit the DCAF1-CRL4 E3 ubiquitin ligase to trigger species-specific SAMHD1 degradation. Determinants of SIV Vpx-mediated primate SAMHD1 degradation have been mapped to its C terminus. In this study, we have identified the N terminus of human SAMHD1 as a major species-specific determinant of Vpx-mediated suppression. The SIVmnd2 and SIVrcm Vpx proteins recognize the N terminus of rhesus, but not human, SAMHD1. We have also demonstrated that variation of two primate lineage-specific residues between human and rhesus SAMHD1 proteins determine resistance to SIVmnd2 and SIVrcm Vpx proteins. These residues (Cys15 and Ser52) are sequentially mutated to Phe in different lineages of Old World monkeys. Consequently, SIVmnd2 and SIVrcm Vpx proteins that could recognize Phe15- and Phe52-containing SAMHD1 could not inactivate human SAMHD1, which contains Cys15 and Ser52. In contrast, SIVmac Vpx, which targets the C terminus of SAMHD1 molecules, could inactivate various primate SAMHD1 molecules with divergent C-terminal sequences. Both C terminus-targeted SIVmac Vpx and N terminus-targeted SIVrcm Vpx require DCAF1 for the induction of SAMHD1 degradation. The ability of SIV Vpx to restrict SAMHD1 among different primate species is a manifestation of the SAMHD1 evolutionary pattern among those species.
Akio Adachi - One of the best experts on this subject based on the ideXlab platform.
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Morphological study on biologically distinct Vpx/vpr mutants of HIV-2.
The journal of medical investigation : JMI, 2020Co-Authors: Ahmad Piroozmand, Mikako Fujita, Akio Adachi, Boonruang Khamsri, Tsuneo UchiyamaAbstract:We have previously shown that human immunodeficiency virus type 2 (HIV-2) without functional Vpx and vpr genes is severely defective for viral growth in lymphocytic cells, and suggested that the virions produced in the absence of Vpx and Vpr are critically damaged. To examine the nature of replication-defect for the Vpx/vpr double mutant, we quantitatively and morphologically studied the virions produced in cells transfected or infected with wild type clone, single (Vpx and vpr mutants) or the double mutant. While no significant difference in virion production was found for various virus clones in transfected cells, a major growth retardation in infected cells was readily observed for the Vpx and Vpx/vpr mutants. In particular, no viral growth was detected for the double mutant. By contrast to the very distinct growth characteristics of the three mutant clones, no appreciable difference in virion morphology was noted. These results indicated that Vpx and Vpr of HIV-2 may cooperatively contribute to virion infectivity without affecting virion morphogenesis.
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Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage.
Frontiers in Microbiology, 2016Co-Authors: Yosuke Sakai, Akio Adachi, Ariko Miyake, Yasuyuki Miyazaki, Hikari Sasada, Masako NomaguchiAbstract:Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution.
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Virological characterization of HIV-2 Vpx gene mutants in various cell systems
Microbes and Infection, 2014Co-Authors: Masako Nomaguchi, Akio AdachiAbstract:Abstract Requirement of intrinsically disordered protein Vpx for HIV-2 replication is cell-type dependent. To define Vpx-dependent conditions, replication ability of HIV-2 Vpx mutants was analyzed in various cell lines that differ in cellular type, differentiation state and/or expression level of anti-HIV-1 SAMHD1 degraded by Vpx. Induction of Vpx-sensitive anti-HIV-2 state was not always associated with SAMHD1 expression. Compared with our previous data in lymphocytic cells, growth-defectiveness of the Vpx mutants in differentiated THP-1 cells, a newly established multi-cycle infection system, was considerably different. Taken together, our results suggest that Vpx plays cell-type dependent role through its undetermined structure and/or function.
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Role of poly-proline motif in HIV-2 Vpx expression
Frontiers in Microbiology, 2014Co-Authors: Ariko Miyake, Mikako Fujita, Masako Nomaguchi, Yasuyuki Miyazaki, Akio AdachiAbstract:Human and simian immunodeficiency viruses (HIV and SIVs) contain several auxiliary genes not found in other retroviruses. These genes are thought to be functionally important for optimal viral replication and persistence in infected individuals. Primate lentiviruses can be classified by the composition of these accessory genes. While viruses of the HIV type1 (HIV-1) group have vif, vpr, vpu, and nef genes, those of the HIV-2 group carry vif, Vpx, vpr, and nef genes (Fujita et al., 2010). Vpx protein encoded by the Vpx gene is unique to non-HIV-1 viruses, and is essential for viral replication in macrophages in contrast to its structural paralog Vpr (Fujita et al., 2010). The most outstanding sequence feature to distinguish Vpx from Vpr is the presence of poly-proline motif (PPM) at its C-terminal region. We have recently shown, by in vitro and in vivo assay systems, that the PPM in HIV-2 Vpx is critical for its efficient translation (Miyake et al., 2014). Although PPM consisting of seven consecutive prolines has been demonstrated to be required for efficient HIV-2 Vpx translation, thereby acquiring viral infectivity in macrophages, the effects of PPM mutations on the degradation of Vpx in cells was not formally analyzed as yet (Fujita et al., 2008; Miyake et al., 2014). Therefore, in this study, we asked whether the PPM plays a role in keeping away from proteasomal and/or lysosomal degradation (Figure (Figure1).1). In order to assess this, we used various expression plasmids for HIV-2 Vpx (pEF-FVpx series) described in a previous study (Miyake et al., 2014): wild-type (WT) plasmid has the Vpx gene derived from HIV-2 GL-AN clone (Kawamura et al., 1994); mutants 103/4A and 106/4A have four consecutive alanine-substitutions at the site of P103-P106 and P106-P109, respectively, and have been shown to express a low/minimum level of mutant Vpx proteins in cells (Figure (Figure1A);1A); a negative control is a frame-shift mutant pEF-FxSt that lacks Vpx expression (ΔVpx). Figure 1 Steady-state levels of various Vpx-PPM mutants in cells as monitored by Western blotting. (A) Structure of the FLAG-tagged HIV-2 GL-AN Vpx construct. Numerals above the schema represent amino acid numbers of the Vpx protein. Positions of lysine and glutamine ... Various expression plasmids were transfected into human 293T cells (Lebkowski et al., 1985) as described before (Adachi et al., 1986), and the amounts of WT and mutant Vpx proteins produced in cells in the absence or presence of a proteasome inhibitor MG-132 (Fujita et al., 2004; McCulley and Ratner, 2012) were comparatively examined by Western blotting (Miyake et al., 2014). A drastic reduction in Vpx expression was observed for mutants 103/4A and 106/4A, 106/4A in particular, both in the absence and presence of MG-132 (Figure (Figure1B).1B). These results showed that neither of these mutants could be rescued with MG-132, suggesting no involvement of the PPM in the proteasome-mediated degradation. Similarly, a lysosome inhibitor Bafilomycin A1 (Yoshimori et al., 1991) did not affect much the level of 103/4A and 106/4A in transfected 293T cells, although a small increase was observed for both mutants (Figure (Figure1B).1B). These results suggested that the low expression level of these PPM mutants may not be attributable to the lysosomal degradation. Proteasomal degradation is generally triggered by the polyubiquitin modification of lysine residues in a protein. There are three lysines in the Vpx of HIV-2 GL-AN clone (Khamsri et al., 2006) (Figure (Figure1A).1A). We generated several clones carrying mutations in these residues. Furthermore, we focused on the 76th glutamine residue (Figure (Figure1A).1A). This amino acid has been reported to interact with DCAF1 for formation of Cullin4-based E3 ubiquitin ligase complex to degrade an anti-HIV restriction factor SAMHD1 (Hrecka et al., 2011; Laguette et al., 2011) by proteasome (Le Rouzic et al., 2007; Srivastava et al., 2008). Mutants K68R, K77R, K84R, and Q76A with or without the 106/4A mutation were constructed as described previously (Miyake et al., 2014) (Figure (Figure1A),1A), and examined for their expression in transfected cells (Figures 1C,D). As shown in Figure Figure1C,1C, only one clone with K84R and 106/4A mutations showed a slight enhancement in agreement with a previous report (Srivastava et al., 2008). Moreover, no significant effect was observed for a mutant carrying Q76A and 106/4A mutations (Figure (Figure1D).1D). These results also suggested that PPM may not be associated with the proteasome-mediated degradation. In total, proteasomal or lysosomal degradation does not account for the extremely low expression level of Vpx exhibited by the PPM mutants. This is consistent with our previous conclusion that PPM is critical for efficient translation of Vpx (Miyake et al., 2014). Molecular mechanism by which PPM enhances Vpx translation to a remarkable extent needs to be determined.
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poly proline motif in hiv 2 Vpx is critical for its efficient translation
Journal of General Virology, 2014Co-Authors: Ariko Miyake, Mikako Fujita, Masami Otsuka, Masako Nomaguchi, Yosuke Sakai, Haruna Fujino, Ryoko Koga, Sogo Kawamura, Yasumasa Iwatani, Akio AdachiAbstract:Human immunodeficiency virus type 2 (HIV-2) carries an accessory protein Vpx that is important for viral replication in natural target cells. In its C-terminal region, there is a highly conserved poly-proline motif (PPM) consisting of seven consecutive prolines, encoded in a poly-pyrimidine tract. We have previously shown that PPM is critical for Vpx expression and viral infectivity. To elucidate the molecular basis underlying this observation, we analysed the expression of Vpx proteins with various PPM mutations by in vivo and in vitro systems. We found that the number and position of consecutive prolines in PPM are important for Vpx expression, and demonstrated that PPM is essential for efficient Vpx translation. Furthermore, mutational analysis to synonymously disrupt the poly-pyrimidine tract suggested that the context of PPM amino acid sequences is required for efficient translation of Vpx. We similarly analysed HIV-1 and HIV-2 Vpr proteins structurally related to HIV-2 Vpx. Expression level of the two Vpr proteins lacking PPM was shown to be much lower relative to that of Vpx, and not meaningfully enhanced by introduction of PPM at the C terminus. Finally, we examined the Vpx of simian immunodeficiency virus from rhesus monkeys (SIVmac), which also has seven consecutive prolines, for PPM-dependent expression. A multi-substitution mutation in the PPM markedly reduced the expression level of SIVmac Vpx. Taken together, it can be concluded that the notable PPM sequence enhances the expression of Vpx proteins from viruses of the HIV-2/SIVmac group at the translational level.
Mikako Fujita - One of the best experts on this subject based on the ideXlab platform.
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Morphological study on biologically distinct Vpx/vpr mutants of HIV-2.
The journal of medical investigation : JMI, 2020Co-Authors: Ahmad Piroozmand, Mikako Fujita, Akio Adachi, Boonruang Khamsri, Tsuneo UchiyamaAbstract:We have previously shown that human immunodeficiency virus type 2 (HIV-2) without functional Vpx and vpr genes is severely defective for viral growth in lymphocytic cells, and suggested that the virions produced in the absence of Vpx and Vpr are critically damaged. To examine the nature of replication-defect for the Vpx/vpr double mutant, we quantitatively and morphologically studied the virions produced in cells transfected or infected with wild type clone, single (Vpx and vpr mutants) or the double mutant. While no significant difference in virion production was found for various virus clones in transfected cells, a major growth retardation in infected cells was readily observed for the Vpx and Vpx/vpr mutants. In particular, no viral growth was detected for the double mutant. By contrast to the very distinct growth characteristics of the three mutant clones, no appreciable difference in virion morphology was noted. These results indicated that Vpx and Vpr of HIV-2 may cooperatively contribute to virion infectivity without affecting virion morphogenesis.
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zinc binding site of human immunodeficiency virus 2 Vpx prevents instability and dysfunction of the protein
Journal of General Virology, 2017Co-Authors: Minami Yamamoto, Masami Otsuka, Haruna Fujino, Ryoko Koga, Kazunori Shimagaki, Halil I Ciftci, Masahiro Kamo, Hiroshi Tateishi, Mikako FujitaAbstract:Human immunodeficiency virus 2 Vpx coordinates zinc through residues H39, H82, C87 and C89. We reported previously that H39, H82 and C87 mutants maintain Vpx activity to facilitate the degradation of SAMHD1. Herein, the expression of Vpx mutants in cells was examined in detail. We demonstrated that the zinc-binding site stabilizes the protein to keep its function in virus growth when low levels of Vpx are expressed. At higher levels of expression, Vpx aggregation could occur, and zinc binding would suppress such aggregation. Among the amino acids involved in zinc coordination, H39 plays the most critical role. In summary, zinc binding appears to mitigate flexibility of the three-helix fold of Vpx, thereby preventing dysfunction.
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mutational analysis of hiv 2 Vpx shows that proline residue 109 in the poly proline motif regulates degradation of samhd1
FEBS Letters, 2015Co-Authors: Halil I Ciftci, Masami Otsuka, Haruna Fujino, Ryoko Koga, Sogo Kawamura, Yasumasa Iwatani, Minami Yamamoto, Hiroshi Tateishi, Mikako FujitaAbstract:In this study, we performed a mutational analysis to determine whether the mechanism by which HIV-2 Vpx confers the capacity for infectivity and viral replication in macrophages is solely dependent on its ability to degrade the host antiviral factor SAMHD1. Contrary to expectations, we demonstrated that P109 in the C-terminal poly-proline motif of HIV-2 Vpx has two unique roles: to facilitate the specific degradation of SAMHD1 in macrophages, and to facilitate multimerization of Vpx, therefore preventing SAMHD1 degradation in the presence of high levels of Vpx.
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Role of poly-proline motif in HIV-2 Vpx expression
Frontiers in Microbiology, 2014Co-Authors: Ariko Miyake, Mikako Fujita, Masako Nomaguchi, Yasuyuki Miyazaki, Akio AdachiAbstract:Human and simian immunodeficiency viruses (HIV and SIVs) contain several auxiliary genes not found in other retroviruses. These genes are thought to be functionally important for optimal viral replication and persistence in infected individuals. Primate lentiviruses can be classified by the composition of these accessory genes. While viruses of the HIV type1 (HIV-1) group have vif, vpr, vpu, and nef genes, those of the HIV-2 group carry vif, Vpx, vpr, and nef genes (Fujita et al., 2010). Vpx protein encoded by the Vpx gene is unique to non-HIV-1 viruses, and is essential for viral replication in macrophages in contrast to its structural paralog Vpr (Fujita et al., 2010). The most outstanding sequence feature to distinguish Vpx from Vpr is the presence of poly-proline motif (PPM) at its C-terminal region. We have recently shown, by in vitro and in vivo assay systems, that the PPM in HIV-2 Vpx is critical for its efficient translation (Miyake et al., 2014). Although PPM consisting of seven consecutive prolines has been demonstrated to be required for efficient HIV-2 Vpx translation, thereby acquiring viral infectivity in macrophages, the effects of PPM mutations on the degradation of Vpx in cells was not formally analyzed as yet (Fujita et al., 2008; Miyake et al., 2014). Therefore, in this study, we asked whether the PPM plays a role in keeping away from proteasomal and/or lysosomal degradation (Figure (Figure1).1). In order to assess this, we used various expression plasmids for HIV-2 Vpx (pEF-FVpx series) described in a previous study (Miyake et al., 2014): wild-type (WT) plasmid has the Vpx gene derived from HIV-2 GL-AN clone (Kawamura et al., 1994); mutants 103/4A and 106/4A have four consecutive alanine-substitutions at the site of P103-P106 and P106-P109, respectively, and have been shown to express a low/minimum level of mutant Vpx proteins in cells (Figure (Figure1A);1A); a negative control is a frame-shift mutant pEF-FxSt that lacks Vpx expression (ΔVpx). Figure 1 Steady-state levels of various Vpx-PPM mutants in cells as monitored by Western blotting. (A) Structure of the FLAG-tagged HIV-2 GL-AN Vpx construct. Numerals above the schema represent amino acid numbers of the Vpx protein. Positions of lysine and glutamine ... Various expression plasmids were transfected into human 293T cells (Lebkowski et al., 1985) as described before (Adachi et al., 1986), and the amounts of WT and mutant Vpx proteins produced in cells in the absence or presence of a proteasome inhibitor MG-132 (Fujita et al., 2004; McCulley and Ratner, 2012) were comparatively examined by Western blotting (Miyake et al., 2014). A drastic reduction in Vpx expression was observed for mutants 103/4A and 106/4A, 106/4A in particular, both in the absence and presence of MG-132 (Figure (Figure1B).1B). These results showed that neither of these mutants could be rescued with MG-132, suggesting no involvement of the PPM in the proteasome-mediated degradation. Similarly, a lysosome inhibitor Bafilomycin A1 (Yoshimori et al., 1991) did not affect much the level of 103/4A and 106/4A in transfected 293T cells, although a small increase was observed for both mutants (Figure (Figure1B).1B). These results suggested that the low expression level of these PPM mutants may not be attributable to the lysosomal degradation. Proteasomal degradation is generally triggered by the polyubiquitin modification of lysine residues in a protein. There are three lysines in the Vpx of HIV-2 GL-AN clone (Khamsri et al., 2006) (Figure (Figure1A).1A). We generated several clones carrying mutations in these residues. Furthermore, we focused on the 76th glutamine residue (Figure (Figure1A).1A). This amino acid has been reported to interact with DCAF1 for formation of Cullin4-based E3 ubiquitin ligase complex to degrade an anti-HIV restriction factor SAMHD1 (Hrecka et al., 2011; Laguette et al., 2011) by proteasome (Le Rouzic et al., 2007; Srivastava et al., 2008). Mutants K68R, K77R, K84R, and Q76A with or without the 106/4A mutation were constructed as described previously (Miyake et al., 2014) (Figure (Figure1A),1A), and examined for their expression in transfected cells (Figures 1C,D). As shown in Figure Figure1C,1C, only one clone with K84R and 106/4A mutations showed a slight enhancement in agreement with a previous report (Srivastava et al., 2008). Moreover, no significant effect was observed for a mutant carrying Q76A and 106/4A mutations (Figure (Figure1D).1D). These results also suggested that PPM may not be associated with the proteasome-mediated degradation. In total, proteasomal or lysosomal degradation does not account for the extremely low expression level of Vpx exhibited by the PPM mutants. This is consistent with our previous conclusion that PPM is critical for efficient translation of Vpx (Miyake et al., 2014). Molecular mechanism by which PPM enhances Vpx translation to a remarkable extent needs to be determined.
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poly proline motif in hiv 2 Vpx is critical for its efficient translation
Journal of General Virology, 2014Co-Authors: Ariko Miyake, Mikako Fujita, Masami Otsuka, Masako Nomaguchi, Yosuke Sakai, Haruna Fujino, Ryoko Koga, Sogo Kawamura, Yasumasa Iwatani, Akio AdachiAbstract:Human immunodeficiency virus type 2 (HIV-2) carries an accessory protein Vpx that is important for viral replication in natural target cells. In its C-terminal region, there is a highly conserved poly-proline motif (PPM) consisting of seven consecutive prolines, encoded in a poly-pyrimidine tract. We have previously shown that PPM is critical for Vpx expression and viral infectivity. To elucidate the molecular basis underlying this observation, we analysed the expression of Vpx proteins with various PPM mutations by in vivo and in vitro systems. We found that the number and position of consecutive prolines in PPM are important for Vpx expression, and demonstrated that PPM is essential for efficient Vpx translation. Furthermore, mutational analysis to synonymously disrupt the poly-pyrimidine tract suggested that the context of PPM amino acid sequences is required for efficient translation of Vpx. We similarly analysed HIV-1 and HIV-2 Vpr proteins structurally related to HIV-2 Vpx. Expression level of the two Vpr proteins lacking PPM was shown to be much lower relative to that of Vpx, and not meaningfully enhanced by introduction of PPM at the C terminus. Finally, we examined the Vpx of simian immunodeficiency virus from rhesus monkeys (SIVmac), which also has seven consecutive prolines, for PPM-dependent expression. A multi-substitution mutation in the PPM markedly reduced the expression level of SIVmac Vpx. Taken together, it can be concluded that the notable PPM sequence enhances the expression of Vpx proteins from viruses of the HIV-2/SIVmac group at the translational level.
Jeremy Luban - One of the best experts on this subject based on the ideXlab platform.
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primate immunodeficiency virus proteins Vpx and vpr counteract transcriptional repression of proviruses by the hush complex
Nature microbiology, 2018Co-Authors: Leonid Yurkovetskiy, Mehmet Hakan Guney, Sean M Mccauley, Ann Dauphin, William E Diehl, Jeremy LubanAbstract:Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, however, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2–4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5–8. A screen for factors that mimic these effects identified the human silencing hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN) and MORC29–13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by Vpx or FAM208A knockdown. Finally, Vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex. The human silencing hub (HUSH) complex represses primate immunodeficiency virus transcription and can be counteracted through degradation mediated by viral Vpx or Vpr proteins.
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Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex
bioRxiv, 2018Co-Authors: Leonid Yurkovetskiy, Mehmet Hakan Guney, Sean M Mccauley, Ann Dauphin, William E Diehl, Jeremy LubanAbstract:Drugs that inhibit HIV-1 replication and prevent progression to AIDS do not eliminate HIV-1 proviruses from the chromosomes of long-lived CD4+ memory T cells. To escape eradication by these antiviral drugs, or by the host immune system, HIV-1 exploits poorly defined host factors that silence provirus transcription. These same factors, though, must be overcome by all retroviruses, including HIV-1 and other primate immunodeficiency viruses, in order to activate provirus transcription and produce new virus. Here we show that Vpx and Vpr, proteins from a wide range of primate immunodeficiency viruses, activate provirus transcription in human CD4+ T cells. Provirus activation required the DCAF1 adaptor that links Vpx and Vpr to the CUL4A/B ubiquitin ligase complex, but did not require degradation of SAMHD1, a well-characterized target of Vpx and Vpr. A loss-of-function screen for transcription silencing factors that mimic the effect of Vpx on provirus silencing identified all components of the Human Silencing Hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN), and MORC2. Vpx associated with the HUSH complex components and decreased steady-state levels of these proteins in a DCAF-dependent manner. Finally, Vpx and FAM208A knockdown accelerated HIV-1 and SIVMAC replication kinetics in CD4+ T cells to a similar extent, and HIV-2 replication required either Vpx or FAM208A disruption. These results demonstrate that the HUSH complex restricts transcription of primate immunodeficiency viruses and thereby contributes to provirus latency. To counteract this restriction and activate provirus expression, primate immunodeficiency viruses encode Vpx and Vpr proteins that degrade HUSH complex components.
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Vpx rescue of hiv 1 from the antiviral state in mature dendritic cells is independent of the intracellular deoxynucleotide concentration
Retrovirology, 2014Co-Authors: Christian Reinhard, Jeremy Luban, Dario BottinelliAbstract:Background: SIVMAC/HIV-2 Vpx recruits the CUL4A-DCAF1 E3 ubiquitin ligase complex to degrade the deoxynucleotide hydrolase SAMHD1. This increases the concentration of deoxynucleotides available for reverse transcription in myeloid cells and resting T cells. Accordingly, transduction of these cells by SIVMAC requires Vpx. Virus-like particles containing SIVMAC Vpx (Vpx-VLPs) also increase the efficiency of HIV-1 transduction in these cells, and rescue transduction by HIV-1, but not SIVMAC, in mature monocyte-derived dendritic cells (MDDCs). Differences in Vpx mechanism noted at that time, along with recent data suggesting that SAMHD1 gains additional restriction capabilities in the presence of type I IFN prompted further examination of the role of Vpx and SAMHD1 in HIV-1 transduction of mature MDDCs. Results: When challenged with Vpx-VLPs, SAMHD1 was degraded in MDDCs even after cells had been matured with LPS, though there was no increase in deoxynucleotide levels. Steady-state levels of HIV-1 late reverse transcription products in mature MDDCs were increased to the same extent by either Vpx-VLPs or exogenous nucleosides. In contrast, only Vpx-VLPs increased the levels of 2-LTR circles and proviral DNA in myeloid cells. These results demonstrate that exogenous nucleosides and Vpx-VLPs both increase the levels of HIV-1 cDNA in myeloid cells, but only Vpx-VLPs rescue 2-LTR circles and proviral DNA in myeloid cells with a previously established antiviral state. Finally, since trans-acting Vpx-VLPs provide long-lasting rescue of HIV-1 vector transduction in the face of the antiviral state, and exogenous nucleosides do not, exogenous nucleosides were used to achieve efficient transduction of MDDCs by vectors that stably encode Vprs and Vpxs from a collection of primate lentiviruses. Vpr from SIVDEB or SIVMUS, Vpx from SIVMAC251 or HIV-2, but not SIVRCM, degraded endogenous SAMHD1, increased steady-state levels of HIV-1 cDNA, and rescued HIV-1 from the antiviral state in MDDCs. Conclusion: Inhibition of deoxynucleotide hydrolysis by promoting SAMHD1 degradation is not the only mechanism by which Vpx rescues HIV-1 in MDDCs from the antiviral state. Vpx has an additional effect on HIV-1 transduction of these cells that occurs after completion of reverse transcription and acts independently of deoxynucleotide levels.
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Vpx rescues hiv 1 transduction of dendritic cells from the antiviral state established by type 1 interferon
Retrovirology, 2011Co-Authors: Thomas Pertel, Christian Reinhard, Jeremy LubanAbstract:Background: Vpx is a virion-associated protein encoded by SIVSM, a lentivirus endemic to the West African sooty mangabey (Cercocebus atys). HIV-2 and SIVMAC, zoonoses resulting from SIVSM transmission to humans or Asian rhesus macaques (Macaca mulatta), also encode Vpx. In myeloid cells, Vpx promotes reverse transcription and transduction by these viruses. This activity correlates with Vpx binding to DCAF1 (VPRBP) and association with the DDB1/RBX1/CUL4A E3 ubiquitin ligase complex. When delivered experimentally to myeloid cells using VSV Gpseudotyped virus-like particles (VLPs), Vpx promotes reverse transcription of retroviruses that do not normally encode Vpx. Results: Here we show that Vpx has the extraordinary ability to completely rescue HIV-1 transduction of human monocyte-derived dendritic cells (MDDCs) from the potent antiviral state established by prior treatment with exogenous type 1 interferon (IFN). The magnitude of rescue was up to 1,000-fold, depending on the blood donor, and was also observed after induction of endogenous IFN and IFN-stimulated genes (ISGs) by LPS, poly(I:C), or poly (dA:dT). The effect was relatively specific in that Vpx-associated suppression of soluble IFN-b production, of mRNA levels for ISGs, or of cell surface markers for MDDC differentiation, was not detected. Vpx did not rescue HIV-2 or SIVMAC transduction from the antiviral state, even in the presence of SIVMAC or HIV-2 VLPs bearing additional Vpx, or in the presence of HIV-1 VLPs bearing all accessory genes. In contrast to the effect of Vpx on transduction of untreated MDDCs, HIV-1 rescue from the antiviral state was not dependent upon Vpx interaction with DCAF1 or on the presence of DCAF1 within the MDDC target cells. Additionally, although Vpx increased the level of HIV-1 reverse transcripts in MDDCs to the same extent whether or not MDDCs were treated with IFN or LPS, Vpx rescued a block specific to the antiviral state that occurred after HIV-1 cDNA penetrated the nucleus.
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characterization of simian immunodeficiency virus sivsm human immunodeficiency virus type 2 Vpx function in human myeloid cells
Journal of Virology, 2008Co-Authors: Thomas Pertel, Jeremy Luban, Caroline Goujon, Vanessa Arfi, Julia LienardAbstract:Human immunodeficiency virus type 2 (HIV-2)/simian immunodeficiency virus SIV SM Vpx is incorporated into virion particles and is thus present during the early steps of infection, when it has been reported to influence the nuclear import of viral DNA. We recently reported that Vpx promoted the accumulation of full-length viral DNA following the infection of human monocyte-derived dendritic cells (DCs). This positive effect was exerted following the infection of DCs with cognate viruses and with retroviruses as divergent as HIV-1, feline immunodeficiency virus, and even murine leukemia virus, leading us to suggest that Vpx counteracted an antiviral restriction present in DCs. Here, we show that Vpx is required, albeit to a different extent, for the infection of all myeloid but not of lymphoid cells, including monocytes, macrophages, and monocytoid THP-1 cells that had been induced to differentiate with phorbol esters. The intracellular localization of Vpx was highly heterogeneous and cell type dependent, since Vpx localized differently in HeLa cells and DCs. Despite these differences, no clear correlation between the functionality of Vpx and its intracellular localization could be drawn. As a first insight into its function, we determined that SIV SM /HIV-2 and SIV RCM Vpx proteins interact with the DCAF1 adaptor of the Cul4-based E3 ubiquitin ligase complex recently described to associate with HIV-1 Vpr and HIV-2 Vpx. However, the functionality of Vpx proteins in the infection of DCs did not strictly correlate with DCAF1 binding, and knockdown experiments failed to reveal a functional role for this association in differentiated THP-1 cells. Lastly, when transferred in the context of a replication-competent viral clone, Vpx was required for replication in DCs.
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Effects of mutant Vpr/Vpx on HIV-2 assembly demonstrated by immunoelectron microscopy.
Proceedings of the National Science Council Republic of China. Part B Life sciences, 2020Co-Authors: Wan Jj, Lee RatnerAbstract:: The virion-associated accessory proteins Vpr and Vpx of human immunodeficiency virus type 2 (HIV-2) are required for efficient viral replication. Vpr could be important for Vpx assembly. To investigate the interaction of Vpr and Vpx with respect to the effects of reverse transcriptase (RT) activity, viral particle information and Vpx expression site directed mutagenesis was carried out to construct Vpr, Vpx and double Vpr/Vpx HIV-2 mutants. These mutants were used for infection of peripheral blood mononuclear cells (PBM), human acute lymphoblastomic leukaemia cells (CEM-CM3) and HeLa CD4+ cells. Visualization of Vpx expression was carried out using FITC and gold labelling by means of laser scanning confocal microscopy and semi quantitative immunoelectron microscopy. Intracellular and extracellular localizations of Vpx were determined by means of fine structural analysis. Up to 80-90% reduction in the RT activity, total number of viral particles, and average Vpx expression was observed after infection of target cells with the Vpr mutant strains. In addition, intracellular Vpx expression was reduced to 51.2% with the Vpr mutant. Only 0.02% Vpx expression was detected after mutation at amino acid 62. These results provide evidence that Vpr or Vpr/Vpx mutants reduce RT activity and interfere with the expression of Vpx in HIV-2 particles during viral assembly. Vpr is efficient for Vpx corporation during viral assembly.
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hiv 2 viral protein x Vpx ubiquitination is dispensable for ubiquitin ligase interaction and effects on macrophage infection
Virology, 2012Co-Authors: Anna Mcculley, Lee RatnerAbstract:HIV-2 Vpx, a virus-associated accessory protein, is critical for infection of non-dividing myeloid cells. To understand the function of Vpx ubiquitination, interaction with an E3 ubiquitin ligase complex, and ability to overcome an inhibition of reverse transcription, we analyzed Vpx lysine mutants for their function and replication capability in macrophages. Both Wt Vpx and Vpx TA (lysine-less Vpx) localized to the cytoplasm and nucleus in HeLa cells. All HIV-2 Vpx lysine mutants were functional in virion packaging. However, ubiquitination was absent with Vpx TA and Vpx K84A mutants, indicating a lack of ubiquitin on positions K68 and K77. Mutants Vpx K68A and K77A were unable to infect macrophages due to impaired reverse transcription from loss of interaction with the ubiquitin substrate receptor, DCAF1. Even though Vpx K84A lacked ubiquitination, it bound DCAF1, and infected macrophages comparable to Wt Vpx.
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hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 Vpx mediated preintegration complex
Journal of Virology, 2008Co-Authors: Xiaogang Cheng, Michael Belshan, Lee RatnerAbstract:Human immunodeficiency virus type 2 (HIV-2) Vpx is required for nuclear translocation of the viral preintegration complex (PIC) in quiescent cells. In order to decipher the mechanism of action of Vpx, a cDNA library was screened with the yeast two-hybrid assay, resulting in the identification of heat shock protein 40, Hsp40/DnaJB6, as a Vpx-interactive protein. Interaction with Vpx was confirmed by glutathione S -transferase (GST) pull-down and coimmunoprecipitation assays. Overexpression of Hsp40/DnaJB6 enhanced Vpx nuclear import, whereas overexpression of a nuclear localization mutant of Hsp40/DnaJB6 (H31Q) or down-regulation of Hsp40/DnaJB6 by small interfering RNA (siRNA) reduced the nuclear import of Vpx. Hsp40/DnaJB6 competed with the Pr55 Gag precursor protein for the binding of Vpx and incorporation into virus-like particles. Overexpression of Hsp40/DnaJB6 promoted viral PIC nuclear import, whereas siRNA down-regulation of Hsp40/DnaJB6 inhibited PIC nuclear import. These results demonstrate a role for Hsp40/DnaJB6 in the regulation of HIV-2 PIC nuclear transport.
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conserved amino acids of the human immunodeficiency virus type 2 Vpx nuclear localization signal are critical for nuclear targeting of the viral preintegration complex in non dividing cells
Virology, 2006Co-Authors: Michael Belshan, Lisa A Mahnke, Lee RatnerAbstract:The HIV-2 viral accessory protein Vpx is related to, but distinct from the Vpr protein of HIV-1. Vpx is packaged into virions and as a component of the viral preintegration complex (PIC) is required for efficient virus replication in non-dividing cells. We have previously reported that the minimal transferable region of Vpx that contained karyophilic properties was aa 65 to 72. Analysis of Vpx sequences from various HIV-2/SIV strains reveals that this region contains highly conserved amino acids, including two basic residues (K68, R70) and three tyrosines (Y66, Y69, Y71). Here, we demonstrate that mutation of the basic or tyrosine residues abolishes PIC nuclear import in arrested cells as assessed by PCR detection of viral integration. Examination of cell-free virus by Western blot indicated that all mutant proteins were incorporated into virions, suggesting that the lack of replication in arrested cells was not due to a loss of Vpx in target cells. Together, these studies map critical residues of the Vpx nuclear localization signal that are required for efficient infection of non-dividing cells.
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identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx
Virology, 2003Co-Authors: Michael Belshan, Lee RatnerAbstract:Abstract The Vpx protein of human immunodeficiency virus type 2 (HIV-2) is a viral accessory protein related to, but distinct from, the Vpr protein of HIV-1. Vpx is packaged into virions and, as a component of the viral preintegration complex (PIC), Vpx is required for efficient virus replication in nondividing cells. Therefore, the localization of Vpx in cells is dynamic and dependent upon discrete domains of the protein. Expressed in the absence of other viral proteins, Vpx localizes to the nucleus of cells. However, if expressed with the Gag protein of HIV-2, Vpx localizes to the plasma membrane of cells. To further understand the regulation of Vpx localization, we fused regions of Vpx to β-galactosidase to identify regions of the protein sufficient to mediate nuclear localization. The minimal transferable region of Vpx that conferred nuclear localization in these assays was aa 65 to 72. Alanine substitution of K68 and R70 in a GFP-Vpx construct abolished nuclear localization, suggesting that the basic residues in this region are important for nuclear import. Analysis of the membrane transport of several GFP-Vpx alanine mutants demonstrated that while separable, the domains of Vpx required for nuclear localization are not distinct from the domains required for membrane transport. The results of heterokaryon shuttling assays indicated that Vpx is not a shuttling protein; however, HIV-2 Vpr did shuttle similar to HIV-1 Vpr.
