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.