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...