RESUMO
After entry into the cell, the early steps of the human immunodeficiency virus type 1 (HIV-1) replication cycle are mediated by two functionally distinct nucleoprotein complexes, the reverse transcription complex (RTC) and preintegration complex (PIC). These two unique viral complexes are responsible for the conversion of the single-stranded RNA genome into double-stranded DNA, transport of the DNA into the nucleus, and integration of the viral DNA into the host cell chromosome. Prior biochemical analyses suggest that these complexes are large and contain multiple undiscovered host cell factors. In this study, functional HIV-1 RTCs and PICs were partially purified by velocity gradient centrifugation and fractionation, concentrated, trypsin digested, and analyzed by LC-MS/MS. A total of seven parallel infected and control biological replicates were completed. Database searches were performed with Proteome Discoverer and a comparison of the HIV-1 samples to parallel uninfected control samples was used to identify unique cellular factors. The analysis produced a total data set of 11055 proteins. Several previously characterized HIV-1 factors were identified, including XRCC6, TFRC, and HSP70. The presence of XRCC6 was confirmed in infected fractions and shown to be associated with HIV-1 DNA by immunoprecipitation-PCR experiments. Overall, the analysis identified 94 proteins unique in the infected fractions and 121 proteins unique to the control fractions with ≥ 2 protein assignments. An additional 54 and 52 were classified as enriched in the infected and control samples, respectively, based on a 3-fold difference in total Proteome Discoverer probability score. The differential expression of several candidate proteins was validated by Western blot analysis. This study contributes additional novel candidate proteins to the growing published bioinformatic data sets of proteins that contribute to HIV-1 replication.
Assuntos
Núcleo Celular/virologia , DNA Viral/genética , HIV-1/genética , Linfócitos/virologia , Nucleoproteínas/genética , Proteoma/genética , Proteínas Virais/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia Líquida , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , HIV-1/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Autoantígeno Ku , Linfócitos/metabolismo , Nucleoproteínas/metabolismo , Ligação Proteica , Proteoma/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transcrição Reversa , Espectrometria de Massas em Tandem , Proteínas Virais/metabolismo , Integração ViralRESUMO
Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein-protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection.
Assuntos
Regulação da Expressão Gênica/imunologia , HIV-1/fisiologia , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Linfócitos T/metabolismo , Linfócitos T/virologia , Linhagem Celular , Humanos , Proteínas Nucleares/genética , Proteoma/genética , TranscriptomaRESUMO
We wanted to determine whether methamphetamine use affects a subset of plasma proteins in HIV-infected persons. Plasma samples from two visits were identified for subjects from four groups: HIV+, ongoing, persistent METH use; HIV+, short-term METH abstinent; HIV+, long term METH abstinence; HIV negative, no history of METH use. Among 390 proteins identified, 28 showed significant changes in expression in the HIV+/persistent METH+ group over the two visits, which were not attributable to HIV itself. These proteins were involved in complement, coagulation pathways and oxidative stress. Continuous METH use is an unstable condition, altering levels of a number of plasma proteins.