Proteomics of HIV-1 Control
Ming Li*, M Lally, and B Ramratnam
Warren Alpert Med Sch, Brown Univ, Providence, RI, US
Background: HIV-1, upon integration into the human genome, becomes for all practical purposes a newly acquired genetic locus. In a minority of infected individuals, viral transcription from this locus is low to absent even in activated CD4+ T lymphocytes that have the capacity to harbor productive infection. Here, we asked whether a transcriptionally quiescent HIV-1 genetic locus was associated with a distinct proteomic signature.
Methods: We recruited individuals with chronic HIV-1 infection with plasma viral load >10,000 (n = 10) or <1000 (n = 10). We used the technique of stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of peripheral blood mononuclear cells isolated from subjects. MS-based differential protein expression patterns were subsequently validated by traditional Western blot. Bioinformatic and wet lab approaches were used to determine the role of identified proteins in the viral life cycle.
Results: We identified >3000 proteins of which 17 were reproducibly under-expressed in individuals with plasma viral load >10,000. An intriguing finding was that 6/17 were histone proteins. We traced this reduction to the relative under-expression of histone-related stem loop binding protein (SLBP) that serves to stabilize histone mRNA. Using HIV-1-infected HeLa T4 and CEM cells, we found that depletion of SLBP led to a ~4-fold increase in HIV-1 DNA integration as determined by Alu-PCR and a 4-fold increase in HIV-1 ms/us RNA as quantified by RT-PCR. SLBP depletion also led to increased retention of known LTR-associated transcription factors such as Spl (4-fold), NF-kB (2-fold), and p300 (6-fold) as determined by ChIP assays using TZM cells with a stably integrated LTR. SLBP depletion led to a more open chromatin structure as identified by nuclease digestion assays.
Conclusions: We identified all classes of histone proteins as being reduced in individuals with plasma viral load >10,000 and traced this reduction to decreased levels of SLBP, a stabilizer of histone mRNA. Our results suggest that lymphocytic cells with decreased SLBP had more open chromatin structures, which, in turn, were associated with enhanced ability of HIV-1 to integrate and higher levels of viral transcriptional activity. Our findings suggest that upon cellular entry, inter-individual differences in chromatin structure may impact cellular viral burden.