Trafficking of HIV‐1 RNA is Mediated by Heterogeneous Nuclear Ribonucleoprotein A2 Expression and Impacts on Viral Assembly

Abstract

These authors contributed equally to this work.Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell. HIV-1 infection is characterized by a lengthy latent period before the onset of acquired immunodeficiency syndrome (AIDS). During this period, abundant viral production is kept in check by the immune system and cells that are killed by infection are replaced. Despite mounting a strong early immune response, HIV-1 expression progressively depletes CD4þ T cells, a situation that leads to a progressive weakening of the immune response to infection and the onset of AIDS (1,2). HIV-1 gene transcription generates a primary 9-kilobase pair (kbp) RNA that has three fates dictated by a tight regulatory circuit and temporal activities of viral proteins. The 9-kbp RNA is multiply spliced following transcription to generate several 2-kbp RNAs that give rise to regulatory proteins Tat, Rev and Nef. Tat accumulates and is primarily responsible for high-level transactivation of the integrated HIV-1 provirus. Once a threshold level of Rev is reached, a molecular switch occurs to promote the inhibition of splicing of the primary transcript. The decreased splicing activity also produces singlyspliced RNA species (4-kbp) (3). Rev binds the Revresponsive cis-acting element RNA (4) to promote the nuclear export of the 9-kbp and singly-spliced 4-kbp HIV-1 RNAs. The 9-kbp RNA is not only a substrate for the translation machinery to generate structural (Gag) and viral enzymes, but in addition, it is selected for encapsidati...

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

“…siRNA duplexes targeting hnRNP A1 and hnRNP A2 sequences (A1-1, 5Ј-AATGGGGAACGCTCACGGACT, and A2-1, 5Ј-AAGCTTTGAAACCACAGAAGA) have been previously described (23,27), as have the Nup62 siRNAs (5Ј-CGGCTTTGCCTTGAATTTAAA and 5Ј-ACCGGC-TTTGCCTTGAATTTA) (28). These and the control nonsilencing siRNA duplexes, referred to here as siNS (5Ј-AATTCTCCGAACGTGTCACGA) were synthesized by Qiagen-Xeragon (Flanders, NJ).…”

“…In experiments in which osmotic stress was induced, the growth medium was replaced with one containing 0.6 M D-sorbitol for 1 h prior to harvesting (37). In studies using LMB, cells were transfected with the bicistronic constructs or pNL4-3, and medium containing 2 nM LMB was added for an additional 12 h prior to cell harvesting (23). In studies using Mnk kinase inhibitor CGP57380, cells were transfected with the bicistronic constructs or pNL4-3, and DMSO or 20 M CGP57380 in DMSO containing medium was added to for an additional 20 h after which cells were processed for IF/FISH, IRES activity, and Western analyses.…”

“…Antibodies, siRNAs, Plasmids, and Reagents-Polyclonal anti-hnRNP A2, anti-hnRNP A1, and pan-specific hnRNP antisera were described earlier (22)(23)(24)(25)(26); sheep anti-Gag p17 and mouse anti-p24 antisera were from Michael Phelan, National Institutes of Health AIDS Reference and Reagent Program). Rabbit anti-p24, mouse anti-GAPDH, mouse anti-pan actin, mab414 (which primarily detects Nup62 as well as other FG repeat containing nucleoporins), goat anti-hnRNP A1, mouse anti-CD81, and rabbit anti-Myc were purchased from Intracell, Techni-Science, Abcam Inc., Cedarlane, Santa Cruz Biotechnology, BD Pharmingen, and US Biological, respectively; rabbit anti-␥-tubulin, rat anti-Nup62, mouse anti-digoxin, and mouse anti-PABP1 were purchased from Sigma-Aldrich.…”

“…Transfections with siRNAs were performed as previously described (4,22,23). Briefly, 6 ϫ 10 5 HeLa cells were seeded per well of 6-well plates for Western analysis and IRES activity determination, whereas 3 ϫ 10 5 cells per well were seeded onto coverslips for imaging studies.…”

“…IF, FISH, Microscopy, and Quantitative Measurements-Immunofluorescence (IF) and fluorescence in situ hybridization (FISH) analyses have been described elsewhere (22,23,40). Microscopy was performed on a Zeiss LSM5 Pascal laser-scanning confocal microscope (Carl-Zeiss) or on a WaveFX/Leica spinning disk confocal microscope equipped with Plan-Apochromat 63ϫ oil immersion objective and an argon/krypton laser.…”