Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA.

hnRNP A2 is a cellular protein that is important for nucleocytoplasmic and cytosolic trafficking of the HIV-1 genomic RNA. Both hnRNP A2's interaction with HIV-1 RNA and its expression levels influence the activities of Rev in mediating nucleocytoplasmic export of the HIV-1 genomic RNA. While the lack of Rev expression during HIV-1 gene expression results in nuclear retention of HIV-1 genomic RNA, we show here by fluorescence in situ hybridization and fractionation studies that the genomic RNA translocates to the cytoplasm when hnRNP A2/B1 are depleted from cells. Polyribosome analyses revealed that the genomic RNA was shunted into a cytoplasmic, dense polyribosomal fraction. This fraction contained several RNA-binding proteins involved in viral gene expression and RNA trafficking but did not contain the translation initiation factor, eIF4G1. Amino acid incorporation into nascent polypeptides in this fraction was also greatly reduced, demonstrating that this fraction contains mRNAs that are poorly translated. These results demonstrate that hnRNP A2/B1 expression plays roles in the nuclear retention of the HIV-1 genomic RNA in the absence of Rev and in the release of the genomic RNA from translationally inactive, cytoplasmic RNP complexes.

Nitration of respiratory epithelial cells by myeloperoxidase depends on extracellular nitrite.

To investigate peroxidase induced 3'-nitrotyrosine (3NT) formation, neutrophil derived myeloperoxidase (MPO) (0.025 microM) was directly administered to A549 epithelial cells with or without H(2)O(2) (150 microM). Little evidence of 3NT was found. In contrast, there was a dose dependent increase in intracellular NO (p<0.001, n=8) following MPO (0.025 microM) treatment, which was further enhanced (p<0.0003, n=8) by addition of H(2)O(2). Extracellular NO also increased after MPO (p<0.002, n=5) and with MPO and H(2)O(2) (p<0.004, n=5). Substantial 3NT formation was only detected following addition of nitrite (NO(2)(-), > or =100 microM), which induced a dose dependent increase in epithelial 3NT. In contrast, protein carbonyl formation and increased GSSG/GSH ratios were associated with MPO treatment even in the absence of NO(2)(-). Co-culture of A549 epithelial cells with polymorphonuclear leukocytes (PMN) (10(6)/ml) led to immunocytochemical detection of epithelial 3NT and induction of nitric oxide synthase (NOS2). However, in a Transwell system direct contact between PMN and A549 cells was necessary for immunodetection of 3NT but not of NOS2 consistent with a role for high local nitrite concentrations. These findings demonstrate dissociation between epithelial endogenous NO production and 3NT formation. Although MPO can influence cellular oxidative stress, particularly in the presence of H(2)O(2), 3NT formation requires the presence of high concentrations of NO(2)(-) in the milieu.

Trafficking of HIV-1 RNA is mediated by heterogeneous nuclear ribonucleoprotein A2 expression and impacts on viral assembly.

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.

A late role for the association of hnRNP A2 with the HIV-1 hnRNP A2 response elements in genomic RNA, Gag, and Vpr localization.

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.