SRP RNA but not SRP protein in HIV-1. (A) RNA from virions and culture media analyzed by a ribonuclease protection assay. HIV-1BRU was propagated in CEM-SS cells obtained through the NIH AIDS Research and Reference Reagent Program. Culture media from uninfected and HIV-1BRU infected cells was concentrated by ultracentrifugation. RNA was extracted using TRIzol (Invitrogen), and it protected both a 150-nucleotide (nt) riboprobe fragment corresponding to sequences from HIV-1 and a 101-nt fragment that hybridized to 7SL RNA. The 344-nt [α-32P] rCTP-labeled riboprobe was similar to previously described chimeric viral/7SL probes (Onafuwa-Nuga et al. 2005). Protected fragments were quantified by adjusting phosphorimager values for the number of C residues in each. Markers are in nucleotides. (B) Nuclease sensitivity of virion-associated RNAs. Concentrated HIV-1 was centrifuged through 6%–18% discontinuous iodixanol gradients (Optiprep, Nycomed Pharma) using a Beckman SW41 Ti rotor at 27,500 rpm for 1 h. Micrococcal nuclease (Takara Biotech) digestion was in either the presence or absence of 0.1% Triton X-100. RNAs from digestions were separated by denaturing PAGE, electrophoretically transferred to nylon membranes (Zeta-Probe GT, BioRad), and UV cross-linked. 7SL RNA was detected with a 32P-labeled probe (5′-TGCTCCGTTTCCGACCTGGGCCGGTTCACCCCTCCTT-3′). Note that in Northern blots, 7SL migrated at ~300 nt. Hybridization to longer products was not detected (not shown), indicating that little if any of the encapsidated 7SL was incorporated as provirus read-through RNA. (C) Western blot analysis for SRP54. Cell or virion lysates prepared in RIPA buffer were cleared of particulates by centrifugation, then boiled and loaded on 12% SDS-PAGE in SDS sample buffer. After electrophoretic transfer to PDVF membranes (BioRad), proteins were detected with monoclonal mouse anti-human SRP54 (BD Biosciences Pharmingen). Antibodies were detected using biotinylated anti-mouse IgG (BRL) and HRP-Streptavidin conjugate (43–4323, Zymed), and visualized using Lumi-Light Substrate (Roche). Markers are in kDa. Note that in addition to SRP54, the monoclonal antibody detected a slower migrating protein in CEM-SS cells. This ~75-kDa species was not detectable in NIH3T3 cells using the same reagents, or by the antibody’s manufacturer in Jurkat cells. It was also not seen in blots of human proteins using less sensitive SRP54-targeted polyclonal antibodies (not shown). Despite this unidentified band, data with CEM-SS cells and the monoclonal antibody are presented here because the relatively high levels of virus production, and good antibody sensitivity with this combination best illustrated the magnitude of SRP protein deficiency in virions.
