The Gammaretroviral p12 protein has multiple domains that function during the early stages of replication

Retrovirology

Kate N Bishop¹  Ian A Taylor²  David J Allen³  Mirella Nader¹  Virginie C Boucherit¹  Darren J Wight¹ 

[1] Division of Virology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK;Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK;Current address: Virus Reference Department, Microbiology Services - Colindale, Health Protection Agency, 61 Colindale Avenue, London, NW9 5EQ, UK

关键词: Chromatin binding;    Post-entry events;    p12;    MLV;    Retrovirus;   

Others  :  1209259
DOI  :  10.1186/1742-4690-9-83

 received in 2012-08-09, accepted in 2012-09-19,  发布年份 2012

【 摘 要 】

Background

The Moloney murine leukaemia virus (Mo-MLV) gag gene encodes three main structural proteins, matrix, capsid and nucleocapsid and a protein called p12. In addition to its role during the late stages of infection, p12 has an essential, but undefined, function during early post-entry events. As these stages of retroviral infection remain poorly understood, we set out to investigate the function of p12.

Results

Examination of the infectivity of Mo-MLV virus-like particles containing a mixture of wild type and mutant p12 revealed that the N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function, and that the N-terminal activity precedes the C-terminal activity in the viral life cycle. By creating a panel of p12 mutants in other gammaretroviruses, we showed that these domains are conserved in this retroviral genus. We also undertook a detailed mutational analysis of each domain, identifying residues essential for function. These data show that different regions of the N-terminal domain are necessary for infectivity in different gammaretroviruses, in stark contrast to the C-terminal domain where the same region is essential for all viruses. Moreover, chimeras between the p12 proteins of Mo-MLV and gibbon ape leukaemia virus revealed that the C-terminal domains are interchangeable whereas the N-terminal domains are not. Finally, we identified potential functions for each domain. We observed that particles with defects in the N-terminus of p12 were unable to abrogate restriction factors, implying that their cores were impaired. We further showed that defects in the C-terminal domain of p12 could be overcome by introducing a chromatin binding motif into the protein.

Conclusions

Based on these data, we propose a model for p12 function where the N-terminus of p12 interacts with, and stabilizes, the viral core, allowing the C-terminus of p12 to tether the preintegration complex to host chromatin during mitosis, facilitating integration.

【 授权许可】

   
2012 Wight et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Goff SP: Host factors exploited by retroviruses. Nat Rev Microbiol 2007, 5:253-263.
• [2]Wolf D, Goff SP: Host restriction factors blocking retroviral replication. Annu Rev Genet 2008, 42:143-163.
• [3]Freed EO: Viral late domains. J Virol 2002, 76:4679-4687.
• [4]Martin-Serrano J, Zang T, Bieniasz PD: Role of ESCRT-I in retroviral budding. J Virol 2003, 77:4794-4804.
• [5]Gottlinger HG, Dorfman T, Sodroski JG, Haseltine WA: Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci U S A 1991, 88:3195-3199.
• [6]Huang M, Orenstein JM, Martin MA, Freed EO: p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease. J Virol 1995, 69:6810-6818.
• [7]Crawford S, Goff SP: Mutations in gag proteins P12 and P15 of Moloney murine leukemia virus block early stages of infection. J Virol 1984, 49:909-917.
• [8]Yuan B, Li X, Goff SP: Mutations altering the moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle. EMBO J 1999, 18:4700-4710.
• [9]Auerbach MR, Shu C, Kaplan A, Singh IR: Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting. Proc Natl Acad Sci U S A 2003, 100:11678-11683.
• [10]Yuan B, Fassati A, Yueh A, Goff SP: Characterization of Moloney Murine Leukemia Virus p12 Mutants Blocked during Early Events of Infection. J Virol 2002, 76:10801-10810.
• [11]Ciuffi A, Bushman FD: Retroviral DNA integration: HIV and the role of LEDGF/p75. Trends Genet 2006, 22:388-395.
• [12]Maertens G, Cherepanov P, Pluymers W, Busschots K, De Clercq E, Debyser Z, Engelborghs Y: LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J Biol Chem 2003, 278:33528-33539.
• [13]Yueh A, Goff SP: Phosphorylated Serine Residues and an Arginine-Rich Domain of the Moloney Murine Leukemia Virus p12 Protein Are Required for Early Events of Viral Infection. J Virol 2003, 77:1820-1829.
• [14]Ikuta K, Luftig RB: Detection of phosphorylated forms of Moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis. J Virol 1988, 62:40-46.
• [15]Dodding MP, Bock M, Yap MW, Stoye JP: Capsid processing requirements for abrogation of Fv1 and Ref1 restriction. J Virol 2005, 79:10571-10577.
• [16]Oshima M, Muriaux D, Mirro J, Nagashima K, Dryden K, Yeager M, Rein A: Effects of Blocking Individual Maturation Cleavages in Murine Leukemia Virus Gag. J Virol 2004, 78:1411-1420.
• [17]Kyere SK, Joseph PRB, Summers MF: The p12 domain is unstructured in a murine leukemia virus p12-CA(N) Gag construct. PLoS One 2008, 3:e1902.
• [18]Nandhagopal N, Simpson AA, Johnson MC, Francisco AB, Schatz GW, Rossmann MG, Vogt VM: Dimeric rous sarcoma virus capsid protein structure relevant to immature Gag assembly. J Mol Biol 2004, 335:275-282.
• [19]Lee S, Nagashima K: Cooperative effect of gag proteins p12 and capsid during early events of murine leukemia virus replication. J Virol 2005, 79:4159-4169.
• [20]Prizan-Ravid A, Elis E, Laham-Karam N, Selig S, Ehrlich M, Bacharach E: The Gag cleavage product, p12, is a functional constituent of the murine leukemia virus pre-integration complex. PLoS Pathog 2010, 6:e1001183.
• [21]Zhang F, Zang T, Wilson SJ, Johnson MC, Bieniasz PD: Clathrin facilitates the morphogenesis of retrovirus particles. PLoS Pathog 2011, 7:e1002119.
• [22]Popov S, Strack B, Sanchez-Merino V, Popova E, Rosin H, Gottlinger HG: Human immunodeficiency virus type 1 and related primate lentiviruses engage clathrin through Gag-Pol or Gag. J Virol 2011, 85:3792-3801.
• [23]Stevens A, Bock M, Ellis S, LeTissier P, Bishop KN, Yap MW, Taylor W, Stoye JP: Retroviral capsid determinants of Fv1 NB and NR tropism. J Virol 2004, 78:9592-9598.
• [24]Mullers E, Stirnnagel K, Kaulfuss S, Lindemann D: Prototype foamy virus gag nuclear localization: a novel pathway among retroviruses. J Virol 2011, 85:9276-9285.
• [25]Tobaly-Tapiero J, Bittoun P, Lehmann-Che J, Delelis O, Giron ML, de The H, Saib A: Chromatin tethering of incoming foamy virus by the structural Gag protein. Traffic 2008, 9:1717-1727.
• [26]De Clercq E: The design of drugs for HIV and HCV. Nat Rev Drug Discov 2007, 6:1001-1018.
• [27]Henderson LE, Gilden RV, Oroszlan S: Amino acid sequence homology between histone H5 and murine leukemia virus phosphoprotein p12. Science 1979, 203:1346-1348.
• [28]Ohkura S, Goldstone DC, Yap MW, Holden-Dye K, Taylor IA, Stoye JP: Novel escape mutants suggest an extensive TRIM5alpha binding site spanning the entire outer surface of the murine leukemia virus capsid protein. PLoS Pathog 2011, 7:e1002011.
• [29]Hulme AE, Perez O, Hope TJ: Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci U S A 2011, 108:9975-9980.
• [30]Perez-Caballero D, Hatziioannou T, Zhang F, Cowan S, Bieniasz PD: Restriction of human immunodeficiency virus type 1 by TRIM-CypA occurs with rapid kinetics and independently of cytoplasmic bodies, ubiquitin, and proteasome activity. J Virol 2005, 79:15567-15572.
• [31]Bock M, Bishop KN, Towers G, Stoye JP: Use of a transient assay for studying the genetic determinants of Fv1 restriction. J Virol 2000, 74:7422-7430.
• [32]Mothes W, Boerger AL, Narayan S, Cunningham JM, Young JA: Retroviral entry mediated by receptor priming and low pH triggering of an envelope glycoprotein. Cell 2000, 103:679-689.
• [33]Yap MW, Nisole S, Lynch C, Stoye JP: Trim5alpha protein restricts both HIV-1 and murine leukemia virus. Proc Natl Acad Sci U S A 2004, 101:10786-10791.
• [34]Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, et al.: Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology 2010, 7:10. BioMed Central Full Text
• [35]Groom HC, Yap MW, Galao RP, Neil SJ, Bishop KN: Susceptibility of xenotropic murine leukemia virus-related virus (XMRV) to retroviral restriction factors. Proc Natl Acad Sci U S A 2010, 107:5166-5171.
• [36]Bruce JW, Bradley KA, Ahlquist P, Young JA: Isolation of cell lines that show novel, murine leukemia virus-specific blocks to early steps of retroviral replication. J Virol 2005, 79:12969-12978.
• [37]Vistica J, Dam J, Balbo A, Yikilmaz E, Mariuzza RA, Rouault TA, Schuck P: Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. Anal Biochem 2004, 326:234-256.