Proteomic Analysis of Early HIV-1 Nucleoprotein Complexes

Abstract

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 11,055 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 three-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.

Cyclophilin B enhances HIV-1 infection

“…CypB has been identified in a number of proteomic studies of HIV infected cells and purified virions (Chertova et al, 2006; Haverland et al, 2014; Schweitzer et al, 2013), including a recent study in which we observed that it was increased in the nuclei of HIV infected Jurkat T-cells (DeBoer et al, 2014). To extend those observations we investigated the subcellular expression of CypB in primary peripheral blood mononuclear cells (PBMCs) during HIV infection (Fig.…”

“…Cyclophilin B (CypB) was identified in a number of proteomic investigations performed by our laboratory, including: a characterization of the interactome of Matrix and Integrase, a study of partially purified preintegration complexes, and a study of the nuclei of HIV-infected T-cells ((DeBoer et al, 2014; Schweitzer et al, 2013; Schweitzer et al, 2012), unpubublished data ). Additionally, CypB was shown to be significantly upregulated in the nuclei of HIV-infected monocyte-derived macrophages (Haverland et al, 2014) and identified as an HIV-dependency factor (Brass et al, 2008).…”

Non-POU Domain-Containing Octamer-Binding Protein Negatively Regulates HIV-1 Infection in CD4+T Cells

“…We initially identified NonO interacting with PICs in HIV-1-infected CD4 + T cell lines, 9 and subsequent studies have also identified NonO to be an HIV-1 interactant. [10][11][12] However, the significance of these interactions and whether NonO plays a crucial role in HIV-1 infection have not been answered. We addressed these questions by investigating the effect of NonO KD on HIV-1 infection and characterized what stages of the replication cycle were affected.…”

“…Proteomic analysis of fractions from HIV-1-infected T cell lines identified NonO as a component of HIV-1 RTC across seven repeat experiments. 10 NonO was also shown to interact with several HIV-1 proteins (including integrase) ectopically expressed in HEK293 and Jurkat cells. 11 Furthermore, NonO was identified in an analysis of the Rev interactome in HeLa cells, and the association between NonO and Rev was enhanced by the presence of the Rev response element.…”

Mass Spectrometry-Based Proteomic Approaches for Discovery of HIV–Host Interactions

“…Among the 25 proteins identified from four replicate experiments, both the eEF1A and eEF1G subunits of eEF1 were identified as functional RT cofactors, providing a basis for studying RT and trafficking of the RTC to the nucleus. In a similar study, proteomic analysis was carried out in RTCs and PICs partially purified by gradient centrifugation fractionation [53]. LC-MS/MS analysis of seven replicates identified 94 cellular proteins unique to the infected fractions.…”