Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe

Cell & Bioscience

Richard Y Zhao²  Ge Li⁴  Zsigmond Benko³  Lin Li¹  Joseph Nkeze⁴ 

[1] AIDS Research Department, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China;Institute of Human Virology, University of Maryland School of Medicine, Baltimore 21201-1192, MD, USA;Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria;Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, Baltimore 21201-1192, MD, USA

关键词: Mammalian cell;    Oxidative stress;    Colony formation;    Cellular proliferation;    Subcellular localization;    Schizosaccharomyces pombe;    Fission yeast;    Functional analysis;    Gene expression;    Viral genome;    HIV-1;   

Others  :  1226111
DOI  :  10.1186/s13578-015-0037-7

 received in 2015-06-09, accepted in 2015-07-27,  发布年份 2015

【 摘 要 】

Background

The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in fission yeast (Schizosaccharomyces pombe).

Results

Each one of the HIV-1 genes was cloned and expressed individually in fission yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between fission yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in fission yeast and mammalian cells. Consistent with the observation in fission yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress.

Conclusions

Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

【 授权许可】

   
2015 Nkeze et al.

【 图 表 】

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【 参考文献 】

• [1]Freed EO. HIV-1 gag proteins: diverse functions in the virus life cycle. Virology. 1998; 251(1):1-15.
• [2]Gottlinger HG. The HIV-1 assembly machine. Aids. 2001; 15 Suppl 5:S13-S20.
• [3]Wilk T, Geiselhart V, Frech M, Fuller SD, Flugel RM, Lochelt M. Specific interaction of a novel foamy virus Env leader protein with the N-terminal Gag domain. J Virol. 2001; 75(17):7995-8007.
• [4]Freed EO, Martin MA. The role of human immunodeficiency virus type 1 envelope glycoproteins in virus infection. J Biol Chem. 1995; 270(41):23883-23886.
• [5]Mammano F, Kondo E, Sodroski J, Bukovsky A, Gottlinger HG. Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains. J Virol. 1995; 69(6):3824-3830.
• [6]Deora A, Spearman P, Ratner L. The N-terminal matrix domain of HIV-1 Gag is sufficient but not necessary for viral protein U-mediated enhancement of particle release through a membrane-targeting mechanism. Virology. 2000; 269(2):305-312.
• [7]Gallay P, Swingler S, Song J, Bushman F, Trono D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell. 1995; 83(4):569-576.
• [8]Accola MA, Strack B, Gottlinger HG. Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J Virol. 2000; 74(12):5395-5402.
• [9]Braaten D, Ansari H, Luban J. The hydrophobic pocket of cyclophilin is the binding site for the human immunodeficiency virus type 1 Gag polyprotein. J Virol. 1997; 71(3):2107-2113.
• [10]Kootstra NA, Munk C, Tonnu N, Landau NR, Verma IM. Abrogation of postentry restriction of HIV-1-based lentiviral vector transduction in simian cells. Proc Natl Acad Sci USA. 2003; 100(3):1298-1303.
• [11]Thali M, Bukovsky A, Kondo E, Rosenwirth B, Walsh CT, Sodroski J et al.. Functional association of cyclophilin A with HIV-1 virions. Nature. 1994; 372(6504):363-365.
• [12]von Schwedler UK, Stemmler TL, Klishko VY, Li S, Albertine KH, Davis DR et al.. Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly. EMBO J. 1998; 17(6):1555-1568.
• [13]Carroll SS, Olsen DB, Bennett CD, Gotlib L, Graham DJ, Condra JH et al.. Inhibition of HIV-1 reverse transcriptase by pyridinone derivatives. Potency, binding characteristics, and effect of template sequence. J Biol Chem. 1993; 268(1):276-281.
• [14]Lu M, Blacklow SC, Kim PS. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol. 1995; 2(12):1075-1082.
• [15]Hill M, Tachedjian G, Mak J. The packaging and maturation of the HIV-1 Pol proteins. Curr HIV Res. 2005; 3(1):73-85.
• [16]Pearl LH, Taylor WR. A structural model for the retroviral proteases. Nature. 1987; 329(6137):351-354.
• [17]Hoffman AD, Banapour B, Levy JA. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology. 1985; 147(2):326-335.
• [18]Oude Essink BB, Das AT, Berkhout B. HIV-1 reverse transcriptase discriminates against non-self tRNA primers. J Mol Biol. 1996; 264(2):243-254.
• [19]Chiu TK, Davies DR. Structure and function of HIV-1 integrase. Curr Top Med Chem. 2004; 4(9):965-977.
• [20]McCune JM, Rabin LB, Feinberg MB, Lieberman M, Kosek JC, Reyes GR et al.. Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell. 1988; 53(1):55-67.
• [21]Weiss CD. HIV-1 gp41: mediator of fusion and target for inhibition. AIDS Rev. 2003; 5(4):214-221.
• [22]Li L, Li HS, Pauza CD, Bukrinsky M, Zhao RY. Roles of HIV-1 auxiliary proteins in viral pathogenesis and host–pathogen interactions. Cell Res. 2005; 15(11–12):923-934.
• [23]Fan L, Peden K. Cell-free transmission of Vif mutants of HIV-1. Virology. 1992; 190(1):19-29.
• [24]Strebel K, Klimkait T, Martin MA. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science. 1988; 241(4870):1221-1223.
• [25]Giga-Hama Y, Kumagai H. Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem. 1999; 30(Pt 3):235-244.
• [26]Piel M, Tran PT. Cell shape and cell division in fission yeast. Curr Biol. 2009; 19(17):R823-R827.
• [27]Zhao Y, Lieberman HB. Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes. DNA Cell Biol. 1995; 14(5):359-371.
• [28]Chino A, Watanabe K, Moriya H. Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe. PLoS One. 2010; 5(3):e9652.
• [29]Lu Q, Bauer JC, Greener A. Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins. Gene. 1997; 200(1–2):135-144.
• [30]Takegawa K, Tohda H, Sasaki M, Idiris A, Ohashi T, Mukaiyama H et al.. Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system. Biotechnol Appl Biochem. 2009; 53(Pt 4):227-235.
• [31]Jacobs E, Gheysen D, Thines D, Francotte M, de Wilde M. The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane. Gene. 1989; 79(1):71-81.
• [32]Toyama R, Bende SM, Dhar R. Transcriptional activity of the human immunodeficiency virus-1 LTR promoter in fission yeast Schizosaccharomyces pombe. Nucleic Acids Res. 1992; 20(10):2591-2596.
• [33]Stutz F, Rosbash M. A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation. EMBO J. 1994; 13(17):4096-4104.
• [34]Zhao Y, Cao J, O'Gorman MR, Yu M, Yogev R. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe. J Virol. 1996; 70(9):5821-5826.
• [35]Zhang C, Rasmussen C, Chang LJ. Cell cycle inhibitory effects of HIV and SIV Vpr and Vpx in the yeast Schizosaccharomyces pombe. Virology. 1997; 230(1):103-112.
• [36]Herrero L, Monroy N, Gonzalez ME. HIV-1 Vpu protein mediates the transport of potassium in Saccharomyces cerevisiae. Biochemistry. 2013; 52(1):171-177.
• [37]Benko Z, Liang D, Agbottah E, Hou J, Chiu K, Yu M et al.. Anti-Vpr activity of a yeast chaperone protein. J Virol. 2004; 78(20):11016-11029.
• [38]Elder RT, Yu M, Chen M, Edelson S, Zhao Y. Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint. Virus Res. 2000; 68(2):161-173.
• [39]Huard S, Chen M, Burdette KE, Fenyvuesvolgyi C, Yu M, Elder RT et al.. HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis. Cell Res. 2008; 18(9):961-973.
• [40]Li G, Park HU, Liang D, Zhao RY. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R. Retrovirology. 2010; 7:59. BioMed Central Full Text
• [41]Iordanskiy S, Zhao Y, Dubrovsky L, Iordanskaya T, Chen M, Liang D et al.. Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R. J Virol. 2004; 78(18):9697-9704.
• [42]Caumont AB, Jamieson GA, Pichuantes S, Nguyen AT, Litvak S, Dupont C. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr Genet. 1996; 29(6):503-510.
• [43]Blanco R, Carrasco L, Ventoso I. Cell killing by HIV-1 protease. J Biol Chem. 2003; 278(2):1086-1093.
• [44]Zhao Y, Elder RT. Yeast perspectives on HIV-1 Vpr. Front Biosci. 2000; 5:905-916.
• [45]Zhao RY, Bukrinsky M, Elder RT. HIV-1 viral protein R (Vpr) and host cellular responses. Indian J Med Res. 2005; 121(4):270-286.
• [46]Li G, Bukrinsky M, Zhao RY. HIV-1 viral protein R (Vpr) and its interactions with host cell. Curr HIV Res. 2009; 7(2):178-183.
• [47]Andreola ML, Litvak S. Yeast and the AIDS virus: the odd couple. J Biomed Biotechnol. 2012; 2012:549020.
• [48]Zhao Y, Elder RT, Chen M, Cao J. Fission yeast expression vectors adapted for positive identification of gene insertion and green fluorescent protein fusion. Biotechniques. 1998;25(3):438–40, 42, 44
• [49]Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993; 123(1):127-130.
• [50]Braddock M, Chambers A, Wilson W, Esnouf MP, Adams SE, Kingsman AJ et al.. HIV-1 TAT “activates” presynthesized RNA in the nucleus. Cell. 1989; 58(2):269-279.
• [51]Ono A. HIV-1 assembly at the plasma membrane: gag trafficking and localization. Future Virol. 2009; 4(3):241-257.
• [52]Zhou W, Resh MD. Differential membrane binding of the human immunodeficiency virus type 1 matrix protein. J Virol. 1996; 70(12):8540-8548.
• [53]Dennin RH, Beyer A. Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV-1 on the surface of HIV-1-infected H9-lymphocytes. J Microsc. 1991; 164(Pt 1):53-60.
• [54]Haffar OK, Popov S, Dubrovsky L, Agostini I, Tang H, Pushkarsky T et al.. Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. J Mol Biol. 2000; 299(2):359-368.
• [55]Salgado GF, Vogel A, Marquant R, Feller SE, Bouaziz S, Alves ID. The role of membranes in the organization of HIV-1 Gag p6 and Vpr: p6 shows high affinity for membrane bilayers which substantially increases the interaction between p6 and Vpr. J Med Chem. 2009; 52(22):7157-7162.
• [56]Ansari-Lari MA, Gibbs RA. Analysis of HIV type 1 reverse transcriptase expression in a human cell line. AIDS Res Hum Retroviruses. 1994; 10(9):1117-1124.
• [57]Levin A, Armon-Omer A, Rosenbluh J, Melamed-Book N, Graessmann A, Waigmann E et al.. Inhibition of HIV-1 integrase nuclear import and replication by a peptide bearing integrase putative nuclear localization signal. Retrovirology. 2009; 6:112. BioMed Central Full Text
• [58]Chen M, Elder RT, Yu M, O'Gorman MG, Selig L, Benarous R et al.. Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast. J Virol. 1999; 73(4):3236-3245.
• [59]Schubert U, Bour S, Ferrer-Montiel AV, Montal M, Maldarell F, Strebel K. The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains. J Virol. 1996; 70(2):809-819.
• [60]Wichroski MJ, Ichiyama K, Rana TM. Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization. J Biol Chem. 2005; 280(9):8387-8396.
• [61]Fackler OT, Kienzle N, Kremmer E, Boese A, Schramm B, Klimkait T et al.. Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization. Eur J Biochem. 1997; 247(3):843-851.
• [62]Boe SO, Bjorndal B, Rosok B, Szilvay AM, Kalland KH. Subcellular localization of human immunodeficiency virus type 1 RNAs, Rev, and the splicing factor SC-35. Virology. 1998; 244(2):473-482.
• [63]Yang Y, Ma J, Song Z, Wu M. HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors. FEBS Lett. 2002; 532(1–2):36-44.
• [64]Zhao Y, Yu M, Chen M, Elder RT, Yamamoto A, Cao J. Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline. Virology. 1998; 246(2):266-276.
• [65]Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990; 265(19):10857-10864.
• [66]Mitchison DO. Physiological and cytological methods for Schizosaccharomyces pombe. Methods Cell Physiol. 1970; 4:131-165.
• [67]Hardy M, Rockenbauer A, Vasquez-Vivar J, Felix C, Lopez M, Srinivasan S et al.. Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap. Chem Res Toxicol. 2007; 20(7):1053-1060.
• [68]Zhou Y, Ratner L. A novel inducible expression system to study transdominant mutants of HIV-1 Vpr. Virology. 2001; 287(1):133-142.
• [69]Frei M. Cell viability and proliferation-trpan blue, in centrifugation. Sigma-Aldrich, St. Louis; 2001.
• [70]Zhao, RY. Interactions of Vpr with host cellular proteins. Reactome. 2006;19. http://dx. doi.org/10.3180/REACT_6288.4 webcite
• [71]Sodroski J, Goh WC, Rosen C, Dayton A, Terwilliger E, Haseltine W. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature. 1986; 321(6068):412-417.
• [72]Pollard VW, Malim MH. The HIV-1 Rev protein. Annu Rev Microbiol. 1998; 52:491-532.
• [73]Malim MH, Bohnlein S, Hauber J, Cullen BR. Functional dissection of the HIV-1 Rev trans-activator—derivation of a trans-dominant repressor of Rev function. Cell. 1989; 58(1):205-214.
• [74]Cochrane AW, Perkins A, Rosen CA. Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function. J Virol. 1990; 64(2):881-885.
• [75]Suhasini M, Reddy TR. Cellular proteins and HIV-1 Rev function. Curr HIV Res. 2009; 7(1):91-100.
• [76]Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989; 338(6212):254-257.
• [77]Stutz F, Neville M, Rosbash M. Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast. Cell. 1995; 82(3):495-506.
• [78]Levin A, Hayouka Z, Friedler A, Loyter A. Over-expression of the HIV-1 Rev promotes death of nondividing eukaryotic cells. Virus Genes. 2010; 40(3):341-346.
• [79]Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993; 75(2):241-251.
• [80]Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS et al.. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science. 1993; 262(5137):1274-1277.
• [81]Fineberg K, Fineberg T, Graessmann A, Luedtke NW, Tor Y, Lixin R et al.. Inhibition of nuclear import mediated by the Rev-arginine rich motif by RNA molecules. Biochemistry. 2003; 42(9):2625-2633.
• [82]No D, Yao TP, Evans RM. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA. 1996; 93(8):3346-3351.
• [83]Thomas HE, Stunnenberg HG, Stewart AF. Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature. 1993; 362(6419):471-475.
• [84]Basi G, Schmid E, Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene. 1993; 123(1):131-136.
• [85]Lewis N, Williams J, Rekosh D, Hammarskjold ML. Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins. J Virol. 1990; 64(4):1690-1697.
• [86]Patki AH, Lederman MM. HIV-1 Tat protein and its inhibitor Ro 24-7429 inhibit lymphocyte proliferation and induce apoptosis in peripheral blood mononuclear cells from healthy donors. Cell Immunol. 1996; 169(1):40-46.
• [87]BXT, Electro Cell Manipulator: ECM 600 protocol 0226. 2001.