Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity
© Abdurahman et al; licensee BioMed Central Ltd. 2007
Received: 20 February 2007
Accepted: 19 March 2007
Published: 19 March 2007
We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N-terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.
The HIV-1 capsid protein (CA, p24) is the building block of the conical core structure of the virus. It is initially produced as a part of the Gag precursor (p55) and during or concomitant with the virus release, p55 is cleaved sequentially into the matrix (MA; p17), capsid, nucleocapsid (NC; p7) and p6 proteins [1, 2]. Capsid protein consists of two independently folded globular domains, the N-and C-terminal domain  connected through a short flexible hinge region.
Several studies have shown that mutations within the gag gene disrupt virus replication or infectivity [4–8] and the infectivity of HIV-1 is critically dependent on proper CA assembly and disassembly following cell entry . Although much of the assembly properties of HIV-1 CA were based on x-ray crystallographic data, NMR and in vitro assembly models, the importance of major homology region , the binding site for cyclophilin A (CypA) [11, 12], and the CA dimer interfaces [13, 14] are some of the functions in CA that have been characterised using mutational analysis.
Surprisingly, although proviral DNAs in H9 cells infected with E98A virus were not detected, a low level of Tat-induced luciferase activity was detected in a single-cell-cycle infectivity assay with TZM-bl cells (Fig. 3B). Given the fact that Tat is critical for the HIV-1 gene expression and reverse transcription [22, 23], we investigated whether a soluble Tat protein released in to the culture supernatant was involved in this assay. To address this issue, possible soluble Tat proteins in the supernatant of transfected HeLa-tat cells was immunoprecipitated using monoclonal antibody against Tat and then tested for the infectivity (Fig. 3C). However, we were unable to inhibit the subtle amount of Tat-induced luciferase activity seen in these cells and subsequently explain this activity. A possible reason may be that Tat is packaged into HIV-1 particles through binding to TAR element [24, 25], although the presence of Tat in virion has never been reported satisfactorily. Consistent with a previous report , we were also unable to detect Tat proteins in Viraffinity concentrated viral lysate using WB analysis with Tat-specific monoclonal antibody.
Since the E98A mutation is located C-terminal to the CypA-binding site and CypA has been suggested to disrupt CA-CA interactions following cell entry of the virus, we tested whether the reason for the diminished viral replication may be due to the lack of CypA incorporation in to the budding particle. However, analysis of virion-associated proteins revealed similar levels of CypA incorporation as in the control virus (Fig. 3D).
List of abbreviations used
human immunodeficiency virus
We would like to thank Ákos Végvari for critical reading and helpful discussions of the manuscript. This work was supported by grants from the Swedish Medical Research Council (grant no. K2000-06X-09501-10B), Swedish International development Cooperation Agency, SIDA (grant no. 2006-0011786) and Tripep AB
- Pettit SC, Moody MD, Wehbie RS, Kaplan AH, Nantermet PV, Klein CA, Swanstrom R: The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions. J Virol. 1994, 68 (12): 8017-8027.PubMed CentralPubMedGoogle Scholar
- Wiegers K, Rutter G, Kottler H, Tessmer U, Hohenberg H, Krausslich HG: Sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual Gag polyprotein cleavage sites. J Virol. 1998, 72: 2846-2854.PubMed CentralPubMedGoogle Scholar
- Monaco-Malbet S, Berthet-Colominas C, Novelli A, Battai N, Piga N, Cheynet V, Mallet F, Cusack S: Mutual conformational adaptations in antigen and antibody upon complex formation between an Fab and HIV-1 capsid protein p24. Structure. 2000, 8 (10): 1069-1077. 10.1016/S0969-2126(00)00507-4.View ArticlePubMedGoogle Scholar
- Abdurahman S, Hoglund S, Goobar-Larsson L, Vahlne A: Selected amino acid substitutions in the C-terminal region of human immunodeficiency virus type 1 capsid protein affect virus assembly and release. J Gen Virol. 2004, 85 (Pt 10): 2903-2913. 10.1099/vir.0.80137-0.View ArticlePubMedGoogle Scholar
- Wang CT, Barklis E: Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants. J Virol. 1993, 67 (7): 4264-4273.PubMed CentralPubMedGoogle Scholar
- von Schwedler UK, Stray KM, Garrus JE, Sundquist WI: Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol. 2003, 77 (9): 5439-5450. 10.1128/JVI.77.9.5439-5450.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Joshi A, Nagashima K, Freed EO: Mutation of Dileucine-Like Motifs in the Human Immunodeficiency Virus Type 1 Capsid Disrupts Virus Assembly, Gag-Gag Interactions, Gag-Membrane Binding, and Virion Maturation. J Virol. 2006, 80 (16): 7939-7951. 10.1128/JVI.00355-06.PubMed CentralView ArticlePubMedGoogle Scholar
- 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 (5): 2545-2552. 10.1128/JVI.78.5.2545-2552.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Forshey BM, von Schwedler U, Sundquist WI, Aiken C: Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol. 2002, 76 (11): 5667-5677. 10.1128/JVI.76.11.5667-5677.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Mammano F, Ohagen A, Hoglund S, Gottlinger HG: Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis. J Virol. 1994, 68 (8): 4927-4936.PubMed CentralPubMedGoogle Scholar
- Kong LB, An D, Ackerson B, Canon J, Rey O, Chen IS, Krogstad P, Stewart PL: Cryoelectron microscopic examination of human immunodeficiency virus type 1 virions with mutations in the cyclophilin A binding loop. J Virol. 1998, 72 (5): 4403-4407.PubMed CentralPubMedGoogle Scholar
- Yoo S, Myszka DG, Yeh C, McMurray M, Hill CP, Sundquist WI: Molecular recognition in the HIV-1 capsid/cyclophilin A complex. J Mol Biol. 1997, 269 (5): 780-795. 10.1006/jmbi.1997.1051.View ArticlePubMedGoogle Scholar
- Gamble TR, Yoo S, Vajdos FF, von Schwedler UK, Worthylake DK, Wang H, McCutcheon JP, Sundquist WI, Hill CP: Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science. 1997, 278 (5339): 849-853. 10.1126/science.278.5339.849.View ArticlePubMedGoogle Scholar
- Sticht J, Humbert M, Findlow S, Bodem J, Muller B, Dietrich U, Werner J, Krausslich HG: A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol. 2005, 12 (8): 671-677. 10.1038/nsmb964.View ArticlePubMedGoogle Scholar
- Gamble TR, Vajdos FF, Yoo S, Worthylake DK, Houseweart M, Sundquist WI, Hill CP: Crystal structure of human cyclophilin A bound to the amino-terminal domain of HIV-1 capsid. Cell. 1996, 87 (7): 1285-1294. 10.1016/S0092-8674(00)81823-1.View ArticlePubMedGoogle Scholar
- Vajdos FF, Yoo S, Houseweart M, Sundquist WI, Hill CP: Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein. Protein Sci. 1997, 6 (11): 2297-2307.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao Y, Chen Y, Schutkowski M, Fischer G, Ke H: Cyclophilin A complexed with a fragment of HIV-1 gag protein: insights into HIV-1 infectious activity. Structure. 1997, 5 (1): 139-146. 10.1016/S0969-2126(97)00172-X.View ArticlePubMedGoogle Scholar
- Chatterji U, Bobardt MD, Stanfield R, Ptak RG, Pallansch LA, Ward PA, Jones MJ, Stoddart CA, Scalfaro P, Dumont JM, Besseghir K, Rosenwirth B, Gallay PA: Naturally Occurring Capsid Substitutions Render HIV-1 Cyclophilin A Independent in Human Cells and TRIM-cyclophilin-resistant in Owl Monkey Cells. J Biol Chem. 2005, 280 (48): 40293-40300. 10.1074/jbc.M506314200.View ArticlePubMedGoogle Scholar
- Sobolev V, Sorokine A, Prilusky J, Abola EE, Edelman M: Automated analysis of interatomic contacts in proteins. Bioinformatics. 1999, 15 (4): 327-332. 10.1093/bioinformatics/15.4.327.View ArticlePubMedGoogle Scholar
- Wei X, Decker JM, Liu H, Zhang Z, Arani RB, Kilby JM, Saag MS, Wu X, Shaw GM, Kappes JC: Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother. 2002, 46 (6): 1896-1905. 10.1128/AAC.46.6.1896-1905.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Guyader M, Kiyokawa E, Abrami L, Turelli P, Trono D: Role for Human Immunodeficiency Virus Type 1 Membrane Cholesterol in Viral Internalization. J Virol. 2002, 76 (20): 10356-10364. 10.1128/JVI.76.20.10356-10364.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Bannwarth S, Gatignol A: HIV-1 TAR RNA: the target of molecular interactions between the virus and its host. Curr HIV Res. 2005, 3 (1): 61-71. 10.2174/1570162052772924.View ArticlePubMedGoogle Scholar
- Jeang KT, Xiao H, Rich EA: Multifaceted Activities of the HIV-1 Transactivator of Transcription, Tat. J Biol Chem. 1999, 274 (41): 28837-28840. 10.1074/jbc.274.41.28837.View ArticlePubMedGoogle Scholar
- Dingwall C, Ernberg I, Gait MJ, Green SM, Heaphy S, Karn J, Lowe AD, Singh M, Skinner MA: HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. Embo J. 1990, 9 (12): 4145-4153.PubMed CentralPubMedGoogle Scholar
- Weeks KM, Crothers DM: RNA recognition by Tat-derived peptides: interaction in the major groove?. Cell. 1991, 66 (3): 577-588. 10.1016/0092-8674(81)90020-9.View ArticlePubMedGoogle Scholar
- Harrich D, Ulich C, Garcia-Martinez LF, Gaynor RB: Tat is required for efficient HIV-1 reverse transcription. Embo J. 1997, 16 (6): 1224-1235. 10.1093/emboj/16.6.1224.PubMed CentralView ArticlePubMedGoogle Scholar
- DeLano WL: The PyMOL Molecular Graphics System. 2002Google Scholar
- Horal P, Svennerholm B, Jeansson S, Rymo L, Hall WW, Vahlne A: Continuous epitopes of the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein and reactivity of human sera to synthetic peptides representing various HIV-1 isolates. J Virol. 1991, 65 (5): 2718-2723.PubMed CentralPubMedGoogle Scholar
- Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985, 230 (4732): 1350-1354. 10.1126/science.2999980.View ArticlePubMedGoogle Scholar
- Jacque JM, Triques K, Stevenson M: Modulation of HIV-1 replication by RNA interference. Nature. 2002, 418 (6896): 435-438. 10.1038/nature00896.View ArticlePubMedGoogle Scholar
- Ou CY, Kwok S, Mitchell SW, Mack DH, Sninsky JJ, Krebs JW, Feorino P, Warfield D, Schochetman G: DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science. 1988, 239 (4837): 295-297. 10.1126/science.3336784.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.