Figure 4

Incorporation of certain RNA- and DNA-binding cellular proteins into HIV-1 viral cores does not correlate with abundance of these proteins in infected cells. A – Western blot detection of the cytoskeleton proteins actin and β tubulin in uninfected Sup-T1, activated and non-activated THP1 cells. Lysates were normalized according to cell counts and then according to the count of β globin DNA using quantitative real-time PCR, and subjected to SDS-PAGE and Western blot analysis. B – Western blot detection of cellular RNA-binding proteins (DHX9, SNRNP200), DNA- binding proteins (MCM5, XRCC5, RUVBL1, RUVBL2), cytoskeleton protein β tubulin, and viral protein CA p24^Gag in the lysates of virus-producing cells (left bands) and in “spin-thru” purified viral cores (right bands). Virus was harvested at 72 h p.i. from Sup-T1, activated and non-activated THP1 cells infected with MLV Env-pseudotyped HIV-1 NL4-3, normalized to CA p24^Gag and subjected to the “spin-thru” core isolation. Lysates of infected cells were normalized according to total protein count and β globin DNA count as described in A. Cellular and viral core preparations were analyzed by Western blotting. C – Quantification of Western blotting results. Western blotting data were quantified using ImageJ software. Results are presented as percentage of the peak value for each protein in the cellular and viral core preparations. D – Quantification of viral genomic RNA in the cores of virions. Viral cores were prepared as described in Figure 1. RNA was isolated from CA p24^Gag-normalized core samples, subjected to reverse transcription with oligo-dT primer and then to quantitative real-time PCR with the primer set specific for positive-strand HIV-1 DNA. The data represents analysis of three independent preparations. Each point shows mean RNA copy number ± SD per 1 ng of p24^CA in the viral core sample.