Vpx is Critical for SIVmne infection of pigtail macaques

Retrovirology

Lee Ratner³  Vanessa Hirsch²  Luis Giavedoni⁵  Vida Hodara⁵  Rajesh Thippeshappa¹  Alison Larsen⁶  Anna McCulley⁴  Xiaogang Cheng⁴  Charles Brown²  Jason T Kimata¹  Michael Belshan⁶ 

[1] Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA;Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA;Division of Oncology, Washington University, Box 8069, 660 S Euclid Ave, St Louis, MO 63110, USA;Division of Molecular Oncology, Washington University School of Medicine, St Louis, MO, USA;Department of Virology and Immunology, Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA;Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, USA

关键词: Macaques;    SIV;    Vpx;   

Others  :  1209313
DOI  :  10.1186/1742-4690-9-32

 received in 2011-07-12, accepted in 2012-04-24,  发布年份 2012

【 摘 要 】

Background

Viral protein X (Vpx) of SIV has been reported to be important for establishing infection in vivo. Vpx has several different activities in vitro, promoting preintegration complex import into the nucleus in quiescent lymphocytes and overcoming a block in reverse transcription in macrophages. Vpx interacts with the DDB1-CUL4-DCAF1 E3 ligase complex, which may or may not be required for the ascribed functions. The goal of the current study was to determine whether these activities of Vpx are important in vivo.

Results

An infectious, pathogenic clone of SIVmne was used to examine correlations between Vpx functions in vitro and in vivo. Three previously described HIV-2 Vpx mutants that were shown to be important for nuclear import of the preintegration complex in quiescent lymphocytes were constructed in SIVmne: A vpx-deleted virus, a truncation of Vpx at amino acid 102 that deletes the C-terminal proline-rich domain (X(102)), and a mutant with tyrosines 66, 69, and 71 changed to alanine (X(y-a)). All mutant viruses replicated similarly to wild type SIVmne027 in primary pigtail macaque PBMCs, and were only slightly retarded in CEMx174 cells. However, all the vpx mutant viruses were defective for replication in both human and pigtail monocyte-derived macrophages. PCR assays demonstrated that the efficiency of reverse transcription and the levels of viral integration in macrophages were substantially reduced for the vpx mutant viruses. In vitro, the X(y-a) mutant, but not the X(102) mutant lost interaction with DCAF1. The wild type SIVmne027 and the three vpx mutant SIVs were inoculated by the intra-rectal route into pigtail macaques. Peak levels of plasma viremia of the vpx mutant SIVs were variable, but consistently lower than that observed in macaques infected with wild type SIVmne. In situ hybridization for SIV demonstrated that compared to wild type SIVmne infected macaques five of the six animals inoculated with the vpx mutant SIVs had only low levels of SIV-expressing cells in the rectum, most intestinal epithelial tissues, spleen, and mesenteric and peripheral nodes.

Conclusions

This work demonstrates that the activities of Vpx to overcome restrictions in culture in vitro are also likely to be important for establishment of infection in vivo and suggest that both the nuclear localization and DCAF1-interaction functions of Vpx are critical in vivo.

【 授权许可】

   
2012 Belshan et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

【 参考文献 】

• [1]Sharp PM, Bailes E, Stevenson M, Emerman M, Hahn BH: Gene acquisition in HIV and SIV. Nature 1996, 383:586-587.
• [2]Tristem M, Marshall C, Karpas A, Petrik J, Hill F: Origin of vpx in lentiviruses. Nature 1990, 347:341-342.
• [3]Tristem M, Purvis A, Quicke DLJ: Complex evolutionary history of primate lentiviral vpr genes. Virology 1998, 240:232-237.
• [4]Cohen EA, Subbramanian RA, Gottlinger HG: Role of auxiliary proteins in retroviral morphogenesis. Curr Top Microbiol Immunol 1996, 214:219-235.
• [5]Kappes JC: Viral protein X. Curr Top Microbiol Immunol 1995, 193:121-132.
• [6]Trono D: When accessories turn out to be essential. Nat Med 1998, 4:1368-1369.
• [7]Mahnke LA, Belshan M, Ratner L: Analysis of HIV-2 Vpx by modeling and insertional mutagenesis. Virology 2006, 348:165-174.
• [8]Pancio H, Ratner L: Human immunodeficiency virus 2 Vpx-Gag interaction. J Virol 1998, 72:5271-5275.
• [9]Fletcher TM, Brichacek B, Sharova N, Newman MA, Stivathis G, Sharp PM, Emerman M, Hahn BH, Stevenson M: Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIVsm. EMBO J 1996, 15:6155-6165.
• [10]Pancio H, Heyden NV, Ratner L: The C-terminal proline-rich tail of HIV-2 Vpx is necessary for nuclear localization of the viral preintegration complex in nondividing cells. J Virol 2000, 74:6162-6167.
• [11]Belshan M, Mahnke LA, Ratner L: 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 2006, 346:118-126.
• [12]Belshan M, Ratner L: Identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx. Virology 2003, 311:7-15.
• [13]Cheng X, Ratner L: Hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 Vpx-mediated preintegration complex. J Virol 2008, 82:1229-1237.
• [14]Mueller SM, Jung R, Weiler S, Lang SM: Vpx proteins of SIVmac239 and HIV-2ROD interact with the cytoskeletal protein alpha-actinin 1. J Gen Virol 2004, 85:3291-3203.
• [15]Singhal PK, Kumar PR, Rao MRKS, Kyasani M, Mahalingam S: Simian imunodeficiency virus Vpx is imported into the nucleus via importin alpha-dependent and -independent pathways. J Virol 2006, 80:526-536.
• [16]Vodicka MA, Koepp DM, Silver PA, Emerman M: HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev 1998, 12:175-185.
• [17]Goujon C, Arfi V, Pertel T, Luban J, Lienard J, Rigal D, Darlix JL, Cimarelli A: Characterization of simian immunodeficiency virus SIVsm/human immunodeficiency virus type 2 Vpx function in human myeloid cells. J Virol 2008, 82:12335-12345.
• [18]Sharova N, Wu Y, Zhu X, Stranska R, Kaushik R, Sharkey M, Stevenson M: Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction. PLoS Pathog 2008, 4:e1000057.
• [19]Srivastava S, Swanson SK, Manel N, Florens L, Washburn MP, Skowronski J: Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog 2008, 4:e100059.
• [20]Fujita M, Otsuka M, Miyoshi M, Khamsri B, Nomaguchi M, Adachi A: Vpx is critical for reverse transcription of the human immunodeficiency virus type 2 genome in macrophages. J Virol 2008, 82:7752-7756.
• [21]LeRouzic E, Belaidouni N, Estrabaud E, Morel M, Rain JC, Transy C, Margottin-Goguet F: HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle 2007, 15:182-188.
• [22]Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S, Florens L, Washburn MP, Skowronski J: Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SamHD1 protein. Nature 2011, 474:658-661.
• [23]Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E, Yatim A, Emiliani S, Schwartz O, Benkirane M: SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 2011, 474:654-657.
• [24]Goldstone DC, Ennis-Adeniran V, Hedden JJ, Groom HC, Rice GI, Christodoulou E, Walker PA, Kelly G, Haire LF, Yap MW, et al.: HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 2011, 480:379-382.
• [25]Powell RD, Holland PJ, Hollis T, Perriono FW: The Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase. Journal of Biological Chemistry 2011, 286:43596-43600.
• [26]Hirsch VM, Sharkey ME, Brown CR, Brichacek B, Goldstein S, Wakefield J, Byrum R, Elkins BH, Lifson JD, Stevenson M: Vpx is required for dissemination and pathogenesis of SIVsmPBj: evidence of macrophage-dependent viral amplification. Nat Med 1998, 4:1401-1408.
• [27]Kimata JT, Kuller L, Anderson DB, Dailey P, Overbaugh J: Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression. Nat Med 1999, 5:535-541.
• [28]Patel PG, YuKimata MT, Biggins JE, Wilson JM, Kimata JT: Highly pathogenic simian immunodeficiency virus mne variants that emerge during the course of infection evolve enhanced infectivity and the ability to downregulate CD4 but not class I major histocompatibility complex antigens. J Virol 2002, 76:6425-6434.
• [29]Kimata JT, Mozaffarian A, Overbaugh J: A lymph node-derived cytopathic simian immunodeficiency virus Mne variant replicates in non-stimulated peripheral blood mononuclear cells. J Virol 1998, 72:245-256.
• [30]Biesinger T, White R, Kimata-Yu MT, Wilson BK, Allan JS, Kimata JT: Relative replication capacity of phenotypic SIV variants during primary infections differs with route of inoculation. Retrovirology 2010, 7:88. BioMed Central Full Text
• [31]Kuller L, Thompson J, Watanabe R, Iskandriati D, Alpers CE, Morton WR, Agy MB: Mucosal antibody expression following rapid SIV(Mne) dissemination in intrarectally infected Macaca nemestrina. AIDS Res Hum Retroviruses 1998, 14:1345-1356.
• [32]Coudedel-Courteille A, Butor C, Jillard V, Guillet J-G, Venet A: Dissemination of SIV after rectal infection preferentially involves paracolic germinal centers. Virology 1999, 260:277-294.
• [33]Gibbs JS, Lackner AA, Lang SM, Simon MA, Sehgal PK, Daniel MD, Desrosiers RC: Progression to AIDS in the absence of a gene for vpr or vpx. J Virol 1995, 69:2378-2383.
• [34]Li Q, Duan L, Estes JD, Ma Z-M, Rourke T, Wang Y, Reilly C, Carlis J, Miller CJ, Haase AT: Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 2005, 434:1148-1152.
• [35]Zhang Z-Q, Schuler T, Zupancic M, Wietgrefe S, Staskus KA, Reimann KA, Reinhart TA, Rogan M, Cavert W, Miller CJ, et al.: Sexual Transmission and Propagation of SIV and HIV in Resting and Activated CD4+ T Cells. Science 1999, 286:1353-1357.
• [36]Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen ISY: HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990, 61:213-222.
• [37]O'Brien WA, Namazi A, Kalhor H, Mao SH, Zack JA, Chen IS: Kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes is slowed by limitations of nucleotide precursors. J Virol 1994, 68:1258-1263.
• [38]McCulley A, Morrow CD: Complementation of human immunodeficiency virus type 1 replication by intracellular selection of Escherichia coli formula supplied in trans. J Virol 2006, 80:9641-9650.
• [39]Ory DS, Neugeboren BA, Mulligan RC: A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci 1996, 93:11400-11406.
• [40]Chackerian B, Haigwood NL, Overbaugh J: Characterization of a CD4-expressing macaque cell line that can detect virus after a single replication cycle and can be infected by diverse simian immunodeficiency virus isolates. Virology 1995, 213:386-394.
• [41]Westervelt P, Trowbridge DB, Epstein LG, Blumberg BM, Li Y, Hahn BH, Shaw GM, Price RW, Ratner L: Macrophage tropism determinant of human immunodeficiency virus type 1 in vivo. J Virol 1992, 66:2577-2582.
• [42]Brown CR, Czapiga M, Kabat J, Dang Q, Ourmanov I, Nishimura Y, Martin MA, Hirsch VM: Unique pathology in simian immunodeficiency virus-infected rapid progressor macaques is consistent with a pathogenesis distinct from that of classical AIDS. J Virol 2007, 81:5594-5606.