Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55Gag , the principal mediator of HIV-1 genomic RNA encapsidation. Gag function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.

SummaryHuman immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.

Staufen1 is a component of transported ribonucleoprotein complexes. Genetic work in Drosophila has suggested that Staufen plays a role in the de-repression of translation of oskar mRNA following localization. To determine whether Staufen1 can play a similar role in mammals, we studied translation of transcripts in the presence or in the absence of Staufen1. Translationally repressed mRNAs were generated by fusing the structured human immunodeficiency virus type 1 trans-activating response (TAR) element to the 5′ end of a reporter transcript. In rabbit reticulocyte lysates and in mammalian cultured cells, the addition of Staufen1 resulted in the up-regulation of reporter activity when translation was driven by the TAR-bearing RNA. In contrast, Staufen1 had no effect on translation of efficiently translated mRNAs lacking an apparent structured 5′ end, suggesting that Staufen1-binding to the 5′ end is required for enhanced translation. Consistently, Staufen1 RNA-binding activity is necessary for this translational effect. In addition, similar up-regulation of translation was observed when Staufen1 was tethered to the 5′ end of mRNAs via other structured RNAs, the highest level of translational increase being obtained with the bona fide Staufen1-binding site of the Arf1 transcript. The expression of Staufen1 promoted polysomal loading of TAR-luciferase transcripts resulting in enhanced translation. Our results support a model in which the expression of Staufen1 and its interaction with the 5′ end of RNA and ribosomes facilitate translation initiation.

Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and Optiprep gradient analyses reveal cosedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By coimmunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1-expressing cells and purified virus. Overexpression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55Gag , viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55Gag . Pr55 Gag synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.

Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1.During the late stages of the HIV-1 3 replication cycle, the full-length HIV-1 viral RNA (vRNA) must be exported from the nucleus and both translated and packaged into new viral particles (1). The orchestration of these events is directed by a diversity of viral and host proteins that interact with each other and with the vRNA to form HIV-1 ribonucleoprotein (RNP) complexes that originate in the nucleus and persist in the cytoplasm (2). Investigations into the composition and functions of the HIV-1 RNP will reveal new information about innate immunity as well as identify new potential therapeutic targets (3-6).The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a predominantly nuclear protein engaged in a number of cellular and viral RNPs for RNA-processing activities, including splicing regulation, nuclear export, microRNA processing, mRNA stability, telomere maintenance, and IRES-mediated translation initiation (7-12). What enables hnRNP A1 to have such broad functions in RNA metabolism is its ability to bind both nuclear and cytoplasmic RNAs (10). In addition to its well characterized role in nuclear RNA processing, hnRNP A1 binds to purine-rich sequences of mRNAs for mRNA turnover and translation (13,14). Close examination of the HIV-1 vRNA reveals many AG-and AU-rich sequences closely resembling hnRNP A1 binding motifs (15). In fact, hnRNP A1 binds to a number of sequences on the HIV-1 vRNA such as the cis-acting repressive sequences, instability elements, and exonic splicing silencer elements, and indeed, hnRNP A1 is implicated in the fate of HIV-1 RNA, including splicing regulation, nucleocytoplasmic export, and cytoplasmic stability (16 -21).Our previous work demonstrated that hnRNP A1 efficiently immunoprecipitated with the HIV-1 vRNA (22) and that siRNA-mediated knockdown of hnRNP A1 caused a dramatic decrease in HIV-1 structural protein, pr55Gag (herein termed Gag) expression and virus production with little effect on steady-state levels of the three HIV-1 RNA species (i.e. 9-, 4-, and 2-kb RNAs) (23). In this work, we show that HIV-1 ...

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...

Our earlier work indicated that the human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) is trafficked to the microtubule-organizing center (MTOC) when heterogeneous nuclear ribonucleoprotein A2/B1 is depleted from cells. Also, Rab7-interacting lysosomal protein promoted dynein motor complex, late endosome and vRNA clustering at the MTOC suggesting that the dynein motor and late endosomes were involved in vRNA trafficking. To investigate the role of the dynein motor in vRNA trafficking, dynein motor function was disrupted by small interference RNA-mediated depletion of the dynein heavy chain or by p50/dynamitin overexpression. These treatments led to a marked relocalization of vRNA and viral structural protein Gag to the cell periphery with late endosomes and a severalfold increase in HIV-1 production. In contrast, rerouting vRNA to the MTOC reduced virus production. vRNA localization depended on Gag membrane association as shown using both myristoylation and Gag nucleocapsid domain proviral mutants. Furthermore, the cytoplasmic localization of vRNA and Gag was not attributable to intracellular or internalized endocytosed virus particles. Our results demonstrate that dynein motor function is important for regulating Gag and vRNA egress on endosomal membranes in the cytoplasm to directly impact on viral production. HIV-12 gene transcription generates a 9-kb primary transcript, the genomic RNA (termed vRNA herein). vRNA can remain unspliced, but it is also singly or multiply spliced to generate over 30 additional mRNAs (1). vRNA encodes 55-and 160-kDa precursor proteins pr55Gag (termed Gag herein) and pr160Gag/Pol that are cleaved following virus assembly during virus maturation to yield both structural proteins and enzymes, which are critical for virus replication. The singly spliced, 4-kb mRNAs encode the auxiliary proteins Vpr, Vpu, and Vif, factors that enhance HIV-1 pathogenesis (2-4) as well as the integral membrane protein envelope (Env) that is found at the surface of virus particles. The multiply spliced, 2-kb mRNAs encode the regulatory proteins Tat, Rev, and Nef, proteins that act at both transcriptional and post-transcriptional levels. Although transcripts that are processed incorrectly are retained and degraded within the nucleus (5, 6), vRNA and the 4-kb mRNAs that both contain intronic sequences are exported from the nucleus to the cytoplasm via Rev and the chromosome region maintenance protein 1 nuclear export pathway (7). The well characterized Rev-mediated RNA transport is accomplished by the concerted activities of host cell factors in the nucleus, nuclear membrane, and in the cytosol (8 -11).It has been largely assumed that following transport of HIV-1 RNAs to the cytoplasm, the RNAs either diffuse to the polysomes for translation and/or diffuse to the plasma membrane for assembly. In fact, the cell cytosol is a cluttered environment requiring regulated and energy-dependent transport processes (Ref. 12, and recently reviewed for HIV-1 (13)). Host cell proteins such as human Rev-intera...