A Late Role for the Association of hnRNP A2 with the HIV-1 hnRNP A2 Response Elements in Genomic RNA, Gag, and Vpr Localization

Abstract

Two cis-acting RNA trafficking sequences (heterogenous ribonucleoprotein A2 (hnRNP A2)-response elements 1 and 2 or A2RE-1 and A2RE-2) have been identified in HIV-1 vpr and gag mRNAs and were found to confer cytoplasmic RNA trafficking in a murine oligodendrocyte assay. Their activities were assessed during HIV-1 proviral gene expression in COS7 cells. Single point mutations that were shown to severely block RNA trafficking were introduced into each of the A2REs. In both cases, this resulted in a marked decrease in hnRNP A2 binding to HIV-1 genomic RNA in whole cell extracts and hnRNP A2-containing polysomes. This also resulted in an accumulation of HIV-1 genomic RNA in the nucleus and a significant reduction in genomic RNA encapsidation levels. Immunofluorescence analyses revealed altered expression patterns for pr55Gag and particularly that for Vpr. Vpr localization became almost completely nuclear and this was reflected in a significant reduction in virion-associated Vpr levels. These effects coincided with late steps of the viral replication cycle and were not seen at early time points post-transfection. Transcription, splicing, steady state RNA levels, and pr55 Gag processing were not affected. On the other hand, viral replication was markedly compromised in A2RE-2 mutant viruses and this correlated with lowered genomic RNA encapsidation levels. These data reveal new insights into the virus-host interactions between hnRNP A2 and the HIV-1 A2REs and their influence on the patterns of HIV-1 gene expression and viral assembly. Human immunodeficiency virus type 1 (HIV-1)1 is the cause of acquired immunodeficiency syndrome (AIDS). Transcription of the integrated provirus produces one primary 9-kb transcript that is spliced to produce three size classes of RNA (1). The smallest size class, the 2-kb RNAs, is constitutively exported to the cytosol early in the HIV-1 replication cycle and encodes for the regulatory proteins Tat, Rev, and Nef. Late in the replication cycle, the two other size classes of RNA, the unspliced, 9-kb genomic RNA and the singly spliced, 4-kb RNAs make their way to the cytosol due principally to the activity of Rev, which binds to the Rev responsive element (RRE) present in these RNAs (2). Whereas an abundant amount of information is available about the mechanisms, cellular cofactors, and regulation involved in Rev-mediated RNA nucleocytoplasmic transport (3), very little is understood about HIV-1 RNA trafficking following Rev's disengagement in the cytosol. Recent work demonstrates a role for the cellular human Rev-interacting protein (hRIP) at this step (4). The HIV-1 structural protein, pr55Gag also plays a role at this late step by binding to RNA via it N-terminal matrix (MA) and C-terminal nucleocapsid (NC) domains (5-7). pr55Gag association to molecular motor proteins (8) provides a mechanism by which RNA trafficking is achieved within the cytoplasm. In support of the existence for a trafficking mechanism are data showing that kinesins and microtubules are both necessary for the traffi...

Biochemical and NMR Study on the Competition between Proteins SC35, SRp40, and Heterogeneous Nuclear Ribonucleoprotein A1 at the HIV-1 Tat Exon 2 Splicing Site

“…It is a multifunctional sequence, because it also belongs to one of the hnRNP A2 protein-target site (37). Binding of protein hnRNP A2 to this site was proposed to be involved in HIV-1 RNA transport (38).…”

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

“…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.…”

“…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.…”

“…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.…”

“…Our previous work demonstrated that hnRNP A1 efficiently immunoprecipitated with the HIV-1 vRNA (22) and that siRNA-mediated knockdown of hnRNP A1 caused a dramatic decrease in HIV-1 structural protein, pr55…”