The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed β-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit β-TrCP, suggesting that this activity is taking place independently from β-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by β-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.

Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb.

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RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription. RNA helicase A (RHA)2 is a member of the DEXH-box (where X can be any amino acid) family of proteins and is also termed DHX9 (1, 2). The DEXH-box proteins, together with the DEAD-box and the Ski2 family members, are referred to as RNA helicases that are able to rearrange the structures of RNA molecules (3). RHA contains a helicase core domain consisting of seven motifs that are conserved for all RNA helicases. Within the N-terminal region of RHA there are two copies of type A double-stranded RNA binding domains. Together with an RGG-box domain located at the C terminus, double-stranded RNA binding domains regulate RNA binding as well as helicase activities of RHA (4). RHA is a nuclear protein and shuttles between the nucleus and the cytoplasm with the assistance of a bidirectional nuclear transport domain consisting of 110 amino acids at the C terminus (5). This function of the RHA nuclear transport domain is subject to regulation of arginine methylation catalyzed by PRMT1 (protein-arginine methyltransferase 1) (6). RHA is able to unwind double-stranded RNA or DNA with the energy derived from hydrolysis of NTPs by virtue of its NTPase activity (1). This property enables RHA to participate in multiple cellular processes from RNA transcription to RNA processing to RNA nuclear export (7). These multiple functions underlie the vital role of RHA in the germ line proliferation and development of Caenorhabditis elegans (8) and also account for the early embryonic lethality observed with RHA knock-out mice (9).The regulation activity of RHA in RNA transcription is implicated by its presence within the RNA polymerase II holoenzyme complex. For example, RHA has been shown to bridge the interactions between RNA polymerase II and transcription co-activators such as CREB-binding protein and BRAC1 (breast cancer-specific tumor suppressor protein 1) (10, 11). RHA also directly interacts with the p65 subunit of NF-B and stimulates NF-B-mediated reporter gene expression (12). Involvement of RHA in transcription is further indicated by the function of its homologue in Drosophila, named the maleless (MLE) gene, that increases gene expression fr...