Proteomic Analysis of
Early HIV‑1 Nucleoprotein
Complexes
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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 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