Novel Staufen1 ribonucleoproteins prevent formation of stress granules but favor encapsidation of HIV-1 genomic RNA

HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital, Montréal, QC, H3T 1E2, Canada.
Journal of Cell Science (Impact Factor: 5.43). 02/2010; 123(Pt 3):369-83. DOI: 10.1242/jcs.055897
Source: PubMed


Human 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.

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    • "FLAG-YFP, FLAG-Cdh1 and FLAG-Cdc20 were generated by polymerase chain reaction (PCR) amplification of pCMV-YFP-topaz (Packard Bioscience/PerkinElmer LifeSciences), HA-Cdh1 and HA-Cdc20 and the resulting fragments were cloned in pFLAG-CMV6a (Sigma) that contains a cytomegalovirus promoter. Plasmids coding for pcDNA3-RSV-Stau155-FLAG and pcDNA3-RSV-Stau1Δ2-FLAG (identified as pcDNA-RSV-Stau1ΔNt88-Flag) and driven by a Rous Sarcoma virus promoter were previously described (36). Mutation of the Stau1 D-box sequence to generate pcDNA3-RSV-Stau1Dmut-FLAG was done by PCR-based site-directed mutagenesis using pcDNA3-RSV-Stau155-FLAG as a template. "
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    ABSTRACT: Staufen1 (Stau1) is a ribonucleic acid (RNA)-binding protein involved in the post-transcriptional regulation of gene expression. Recent studies indicate that Stau1-bound messenger RNAs (mRNAs) mainly code for proteins involved in transcription and cell cycle control. Consistently, we report here that Stau1 abundance fluctuates through the cell cycle in HCT116 and U2OS cells: it is high from the S phase to the onset of mitosis and rapidly decreases as cells transit through mitosis. Stau1 down-regulation is mediated by the ubiquitin-proteasome system and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Stau1 interacts with the APC/C co-activators Cdh1 and Cdc20 via its first 88 N-terminal amino acids. The importance of controlling Stau155 levels is underscored by the observation that its overexpression affects mitosis entry and impairs proliferation of transformed cells. Microarray analyses identified 275 Stau155-bound mRNAs in prometaphase cells, an early mitotic step that just precedes Stau1 degradation. Interestingly, several of these mRNAs are more abundant in Stau155-containing complexes in cells arrested in prometaphase than in asynchronous cells. Our results point out for the first time to the possibility that Stau1 participates in a mechanism of post-transcriptional regulation of gene expression that is linked to cell cycle progression in cancer cells.
    Nucleic Acids Research 06/2014; 42(12). DOI:10.1093/nar/gku506 · 9.11 Impact Factor
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    • "Both the Staufen homologs are reported to form RNA-protein complexes and participate in transport, stability and decay of mRNA in human neurons or other polarized cells thereby regulating the spatial and the temporal gene expressions [30,39]. Recently, it was deciphered that Staufen 1, despite being a host protein, is selectively packaged in HIV-1 infectious particle through its interaction with HIV-1 Gag [34,35], while there were no previous reports of hStau-2 being involved in any phase of the HIV-1 life cycle. It was intriguing to identify hStau-2 in the pull-down samples of Rev as we noticed that Rev, a protein from the pathogen and hStau-2, a protein from the host, had overlapping biochemical and functional properties. "
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    ABSTRACT: The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell. In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines. With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.
    Retrovirology 02/2014; 11(1):18. DOI:10.1186/1742-4690-11-18 · 4.19 Impact Factor
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    • "Staufen1 was used as another dsRBP that has a positive activity on the virus by binding to Gag and by increasing translation from TAR-containing RNAs [9,76]. In agreement with its PKR-independent mechanism on translation [9], we found that Staufen1 did not inhibit PKR activation supporting a combined inhibition of PKR by TRBP, ADAR1 and PACT and a different mechanism for Staufen1 via Gag multimerization, HIV-1 assembly and encapsidation of genomic RNA [77-79], all contributing to viral replication. Further studies will determine if PKR forms a different protein complex at the beginning of HIV-1 infection when PKR and eIF2α are activated, if PACT has a different activity in this context and how it may contribute to the pathogenicity induced by the virus. "
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    ABSTRACT: HIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2alpha), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified. PKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293 T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2alpha phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2alpha phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs. In contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.
    Retrovirology 09/2013; 10(1):96. DOI:10.1186/1742-4690-10-96 · 4.19 Impact Factor
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