RESUMO
During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by approximately 98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2(-/-) mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.
Assuntos
Anexina A2/fisiologia , HIV-1/fisiologia , HIV-1/patogenicidade , Animais , Anexina A2/antagonistas & inibidores , Anexina A2/deficiência , Anexina A2/genética , Sequência de Bases , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Células Jurkat , Macrófagos/fisiologia , Macrófagos/virologia , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Proteínas S100/fisiologia , Vírion/fisiologia , Virulência/fisiologia , Montagem de Vírus/fisiologia , Liberação de Vírus/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Avian sarcoma and leukosis virus subgroup A (ASLV-A) entry is mediated by interactions between the viral glycoprotein EnvA and its cognate receptor Tva. Previously, some interesting mutants of ASLV-A have been selected by others which can use chicken Tva, but not quail Tva, for efficient entry. The mutant phenotypes are caused by two point mutations within the surface subunit of EnvA (S. L. Holmen, D. C. Melder, and M. J. Federspiel, J. Virol. 75:726-737, 2001). In this study, we have shown that the altered receptor specificity maps to the LDL-A module of Tva. Further, we have identified two residues in the chicken LDL-A module that allow more efficient viral entry by the mutant viruses. These results demonstrate that the altered receptor specificity of the mutant viruses is determined by specific interactions with residues in the LDL-A module of Tva.
Assuntos
Aminoácidos/metabolismo , Vírus da Leucose Aviária/metabolismo , Proteínas Aviárias/metabolismo , Vírus do Sarcoma Aviário/metabolismo , Receptores Virais/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Galinhas , Interações Hidrofóbicas e Hidrofílicas , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genéticaRESUMO
Rong et al. have demonstrated previously that with a few substitutions, the fourth repeat of human low-density lipoprotein (hLDL-A4) receptor can functionally replace the LDL-A module of Tva, the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), in viral entry (L. Rong, K. Gendron, and P. Bates, Proc. Natl. Acad. Sci. USA 95:8467-8472, 1998). Here we have shown that swapping the amino terminus of hLDL repeat 5 (hLDL-A5) with that of Tva, in addition to the corresponding substitutions made in human LDL-A4, was required to convert hLDL-A5 into an efficient ASLV-A receptor. These results substantiated our previous findings regarding the role of the specific residues in the viral interaction domain of Tva and demonstrated the critical role of the amino terminus of the Tva LDL-A module in ASLV-A infection. Furthermore, we have shown that the residues between cysteines 2 and 3 of the Tva LDL-A module in a Tva/LDL-A5 chimeric protein can be functionally replaced by the corresponding region of another LDL-A module, human LDL receptor-related protein repeat 22 (LDL-A22), to mediate efficient ASLV-A entry. Since the only conserved feature between the C2-C3 region of LDL-A22 and the Tva LDL-A module is that both contain nine amino acids of which none are conserved, we conclude that the spacing between C2 and C3 of the LDL-A module of Tva is an important determinant for ASLV-A entry. Thus, the present study provides strong evidence to support our hypothesis that one role of the N terminus of the LDL-A module of Tva is to allow proper folding and conformation of the protein for optimal interaction with the viral glycoprotein EnvA in ASLV-A entry.