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 pr55Gag 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.
Highlights
C Supported by studentships from the Fondation Georges-Phenix and Fonds pour la recherche en santedu Quebec (FRSQ)
We found a significant reduction in genomic RNA in the hnRNP A2 immunoprecipitate (by 35% in A2RE-1 A8G and by more than 80% in A2RE-2 A8G (Fig. 2E); there was no more than a 5% variation in three independent experiments)
These data demonstrate that the specific point mutations introduced in each of the A2REs resulted in lowered hnRNP A2 binding while, in contrast, the association of hnRNP A1 to Human immunodeficiency virus type 1 (HIV-1) RNA was not affected by these introduced mutations
Summary
DNA Proviral Constructs—A2RE-1 and -2 are located at nt 1192– 1213 and nt 6157– 6178 in HxBc2-based proviral DNA, HxBru [39], respectively. Equal quantities of hnRNP A2-containing polysomes were subsequently immunoprecipitated using a mouse hnRNP A2 antiserum (EF67) [44], and the purified RNA was used in RT-PCR analysis for total and genomic HIV-1 RNA, as described above. Total RNA was extracted using TRIzol LS Reagent (Invitrogen) from cells at 36 – 40-h post-transfection, followed by Northern blotting using a [32P]dCTP-labeled cDNA probe to the HIV-1 untranslated region [25, 39, 46]. The inclusion of two ABS in intronic sequences promotes distal 5Ј-splice site utilization because of the binding hnRNP A1 on these elements For both in vitro assays, radiolabeled pre-mRNAs were prepared by in vitro transcription in the presence of tri-methyl cap analogue and [32P]UTP (1000 Ci/mmol; ICN), gel purified, and used in both types of in vitro splicing reactions at 15,000 cpm per reaction at 30 °C for 2 h exactly as described [54].
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