INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo

Retrovirology

Ganjam V Kalpana²  Christopher Aiken¹  Vinayaka R Prasad²  Vaibhav B Shah¹  Achintya Pal³  Xuhong Wu³  Minh Nguyen³  Sheeba Mathew³ 

[1] Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, A-5301 Medical Center North, Nashville, TN, 37232-2363, USA;Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461, USA;Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, 10461, USA

关键词: Integration;    Reverse transcription;    Morphogenesis;    Host factors;    HIV-1;    IN;    INI1;   

Others  :  1209111
DOI  :  10.1186/1742-4690-10-66

 received in 2013-04-03, accepted in 2013-06-05,  发布年份 2013

【 摘 要 】

Background

Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication.

Results

A reverse yeast two-hybrid system was used to identify

I

NI1-

i

nteraction

d

efective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1_NL4-3 and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro.

Conclusions

Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.

【 授权许可】

   
2013 Mathew et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Adamson CS, Freed EO: Human immunodeficiency virus type 1 assembly, release, and maturation. Adv Pharmacol 2007, 55:347-387.
• [2]Ganser-Pornillos BK, von Schwedler UK, Stray KM, Aiken C, Sundquist WI: Assembly properties of the human immunodeficiency virus type 1 CA protein. J Virol 2004, 78:2545-2552.
• [3]Carter GC, Bernstone L, Baskaran D, James W: HIV-1 infects macrophages by exploiting an endocytic route dependent on dynamin, Rac1 and Pak1. Virology 2011, 409:234-250.
• [4]Brown P: Integration. In Retroviruses. Edited by Coffin JM, Hughes SH, Varmus HE. Cold Spring harbor Laboratory Press; 1997:161-203.
• [5]Briones MS, Chow SA: A new functional role of HIV-1 integrase during uncoating of the viral core. Immunol Res 2010, 48:14-26.
• [6]Bukovsky A, Gottlinger H: Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J Virol 1996, 70:6820-6825.
• [7]Engelman A: In vivo analysis of retroviral integrase structure and function. Adv Virus Res 1999, 52:411-426.
• [8]Engleman A, Englund G, Orenstein JM, Martin MA, Craigie R: Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol 1995, 69:2729-2736.
• [9]Gallay P, Hope T, Chin D, Trono D: HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway. Proc Natl Acad Sci U S A 1997, 94:9825-9830.
• [10]Leavitt AD, Robles G, Alesandro N, Varmus HE: Human Immunodeficiency Virus Type 1 Integrase Mutants Retain In Vitro Integrase Activity yet Fail to Integrase Viral DNA Efficiently during Infection. J Virol 1996, 70:721-728.
• [11]Lu R, Limon A, Devroe E, Silver PA, Cherepanov P, Engelman A: Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication. J Virol 2004, 78:12735-12746.
• [12]Shin C-G, Taddeo B, Haseltine WA, Farnet CM: Genetic analysis of the human immunodeficiency virus type1 integrase protein. J Virol 1994, 64:2421-2425.
• [13]Taddeo B, Haseltine WA, Farnet CM: Integrase mutants of human immunodeficiency virus type 1 with a specific defect in integration. J Virol 1994, 68:8401-8405.
• [14]Wiskerchen M, Muesing MA: Human immunodeficiency virus type 1 integrase: Effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. J Virol 1995, 69:376-386.
• [15]Wiskerchen M, Muesing MA: Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene. J Virol 1995, 69:597-601.
• [16]Wapling J, Moore KL, Sonza S, Mak J, Tachedjian G: Mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer. J Virol 2005, 79:10247-10257.
• [17]Chiang CC, Wang SM, Tseng YT, Huang KJ, Wang CT: Mutations at human immunodeficiency virus type 1 reverse transcriptase tryptophan repeat motif attenuate the inhibitory effect of efavirenz on virus production. Virology 2009, 383:261-270.
• [18]Chiang CC, Wang SM, Pan YY, Huang KJ, Wang CT: A single amino acid substitution in HIV-1 reverse transcriptase significantly reduces virion release. J Virol 2010, 84:976-982.
• [19]König R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT, Chiang CY, Tu BP, De Jesus PD, Lilley CE, et al.: Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 2008, 135:49-60.
• [20]Kalpana GV, Marmon S, Wang W, Crabtree GR, Goff SP: Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5 [see comments]. Science 1994, 266:2002-2006.
• [21]Yung E, Sorin M, Pal A, Craig E, Morozov A, Delattre O, Kappes J, Ott D, Kalpana GV: Inhibition of HIV-1 virion production by a transdominant mutant of Integrase interactor 1. Nat Med 2001, 7:920-926.
• [22]Yung E, Sorin M, Wang EJ, Perumal S, Ott D, Kalpana GV: Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance. J Virol 2004, 78:2222-2231.
• [23]Morozov A, Yung E, Kalpana G: Structure-function analysis of integrase interactor 1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly conserved region. Proc Natl Acad Sci USA 1998, 95:1120-1125.
• [24]Craig E, Zhang ZK, Davies KP, Kalpana GV: A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis. EMBO J 2002, 21:31-42.
• [25]Cano J, Kalpana GV: Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6. J Virol 2011, 85:2254-2265.
• [26]Sorin M, Yung E, Wu X, Kalpana GV: HIV-1 replication in cell lines harboring INI1/hSNF5 mutations. Retrovirology 2006, 3:56. BioMed Central Full Text
• [27]Miller MD, Bushman FD: HIV integration. Ini1 for integration? Curr Biol 1995, 5:368-370.
• [28]Lesbats P, Botbol Y, Chevereau G, Vaillant C, Calmels C, Arneodo A, Andreola ML, Lavigne M, Parissi V: Functional Coupling between HIV-1 Integrase and the SWI/SNF Chromatin Remodeling Complex for Efficient in vitro Integration into Stable Nucleosomes. PLoS Pathog 2011, 7:e1001280.
• [29]Sorin M, Cano J, Das S, Mathew S, Wu X, Davies KP, Shi X, Cheng SW, Ott D, Kalpana GV: Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication. PLoS Pathog 2009, 5:e1000463.
• [30]Das S, Kalpana GV: Reverse two-hybrid screening to analyze protein-protein interaction of HIV-1 viral and cellular proteins. Methods Mol Biol 2009, 485:271-293.
• [31]Maroun M, Delelis O, Coadou G, Bader T, Segeral E, Mbemba G, Petit C, Sonigo P, Rain JC, Mouscadet JF, et al.: Inhibition of early steps of HIV-1 replication by SNF5/Ini1. J Biol Chem 2006, 281:22736-22743.
• [32]Krissinel E, Henrick K: Inference of macromolecular assemblies from crystalline state. J Mol Biol 2007, 372:774-797.
• [33]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, 79:71-81.
• [34]Wang JY, Ling H, Yang W, Craigie R: Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein. EMBO J 2001, 20:7333-7343.
• [35]Hehl EA, Joshi P, Kalpana GV, Prasad VR: Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins. J Virol 2004, 78:5056-5067.
• [36]Wu X, Liu H, Xiao H, Conway JA, Hehl E, Kalpana GV, Prasad V, Kappes JC: Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex. J Virol 1999, 73:2126-2135.
• [37]Shah VB, Aiken C: In vitro uncoating of HIV-1 cores. J Vis Exp 2011, 57:e3384.
• [38]Cherepanov P, Maertens G, Proost P, Devreese B, Van Beeumen J, Engelborghs Y, De Clercq E, Debyser Z: HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J Biol Chem 2003, 278:372-381.
• [39]Hamamoto S, Nishitsuji H, Amagasa T, Kannagi M, Masuda T: Identification of a novel human immunodeficiency virus type 1 integrase interactor, Gemin2, that facilitates efficient viral cDNA synthesis in vivo. J Virol 2006, 80:5670-5677.
• [40]Jiang J, Ablan SD, Derebail S, Hercik K, Soheilian F, Thomas JA, Tang S, Hewlett I, Nagashima K, Gorelick RJ, et al.: The interdomain linker region of HIV-1 capsid protein is a critical determinant of proper core assembly and stability. Virology 2011, 421:253-265.
• [41]Yufenyuy EL, Aiken C: The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid. Retrovirology 2013, 10:29. BioMed Central Full Text
• [42]Iordanskiy S, Berro R, Altieri M, Kashanchi F, Bukrinsky M: Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin. Retrovirology 2006, 3:4. BioMed Central Full Text
• [43]Das S, Cano J, Kalpana GV: Multimerization and DNA binding properties of INI1/hSNF5 and its functional significance. J Biol Chem 2009, 284:19903-19914.
• [44]Kalpana GV, Goff SP: Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system. Proc Natl Acad Sci USA 1993, 90:10593-10597.
• [45]Butler SL, Hansen MS, Bushman FD: A quantitative assay for HIV DNA integration in vivo. Nat Med 2001, 7:631-634.
• [46]Liszewski MK, Yu JJ, O'Doherty U: Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods 2009, 47:254-260.