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

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.Fil: Abrahamyan, Levon G.. Davis Jewish General Hospital; CanadáFil: Chatel Chaix, Laurent. Davis Jewish General Hospital; CanadáFil: Ajamian, Lara. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Milev, Miroslav P.. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Monette, Anne. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Clément, Jean François. Davis Jewish General Hospital; CanadáFil: Song, Rujun. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Lehmann, Martin. Davis Jewish General Hospital; CanadáFil: DesGroseillers, Luc. University Of Montreal; CanadáFil: Laughrea, Michael. Mc Gill University; Canadá. Davis Jewish General Hospital; CanadáFil: Boccaccio, Graciela Lidia. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Mouland, Andrew J.. Mc Gill University; Canadá. Davis Jewish General Hospital; Canad

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular dystrophy

Abstract Background Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. Case presentation Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual. Conclusion Recessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy

Differential effects of hnRNP D/AUF1 isoforms on HIV-1 gene expression

Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42

Characterizing the staufen 1 human immunodeficiency virus type 1 ribonucleoprotein

Human Immunodeficiency Virus type 1 (HIV-1) is a member of the Retrovirus family and is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). As an obligate intracellular parasite, HIV-1 uses cellular proteins and machinery to ensure transmission to uninfected cells. One such cellular protein involved in the late stages of viral replication is Staufen1. It is a main component of mRNA ribonucleoproteins (RNPs) and plays distinct roles in mRNA transport, translation and decay. In the context of HIV-1, Staufen1 was initially found to be selectively encapsidated into the virions. Subsequent work determined its association with vRNA and precursor protein Gag within HIV-1 RNPs. Recently we demonstrated that Staufen1 regulates the viral assembly process by inducing Gag multimerization.In Chapter 2 of the present work, we used tandem affinity purification method followed by mass spectrometry to characterize the Staufen1-containing RNPs. We focused on the investigation of the proteins that associate with Staufen1 complexes, isolated from cells that either do or do not express HIV-1. We further performed a detailed comparison of the protein content between native Staufen1 and Staufen1 HIV-1 RNPs and defined the modulations caused from HIV-1 expression. In Chapter 3, we used bimolecular and trimolecular fluorescence complementation methods (BiFC and TriFC) that allow for direct visualization and localization of protein-protein and protein-protein and RNA interactions in living cells, to show that Gag and Staufen1 interact, which was demonstrated by small multi-fluorescent punctae or vesicles in cells. We demonstrated that these protein partners mainly associate in the cytoplasm. However, we also found that they interact at cholesterol-enriched GM-1-containing membrane microdomains. Importantly, Gag specifically recruited Staufen1 to these lipid raft membranes suggesting a key function for this host factor during Gag assembly. Notably in TriFC experiments, in which one protein partner was tethered to mRNA, Gag-Staufen1 interactions were observed demonstrating active recruitment of one protein when the other is bound to mRNA.Overall, we used both proteomics and live cell visualization to examine fundamental viral-host interactions. We have completely characterized the composition of Staufen1 RNPs and have demonstrated how HIV-1 uses these RNPs for its own purpose. These studies also enhance our understanding of the mechanisms and the specific dynamic features of the viral-host (Gag-Staufen1) interactions in living cells, as monitored by the modern fluorescence complementation assays. The findings presented here are significant help to advance research in the HIV-1 field because a better understanding of the mechanism of retrovirus assembly and budding will aid in the development of novel antiviral therapies targeting the critical late steps in the viral replication cycle.Le virus d'immunodéficience humaine de type 1 (VIH-1) est un membre de la famille de Rétrovirus et est responsable du syndrome d'immunodéficience acquise (SIDA). Considéré comme un parasite intracellulaire obligatoire, le VIH-1 utilise les protéines et les machineries cellulaires pour assurer la transmission aux cellules non infectées. Un des facteurs impliqués dans les étapes finales de réplication virale est la protéine Staufen1. Cette protéine est un composant important des ARNm ribonucléoprotéiques et joue des rôles clés dans le transport, la traduction et la dégradation de l'ARNm. Concernant le VIH-1, Staufen1 a été initialement découvert comme étant spécifiquement encapsidé dans les virions. Des travaux plus approfondis ont déterminé son association avec l'ARNv et le précurseur Gag dans les VIH-1 RNPs. Récemment, nous avons démontré que Staufen1 régule le processus d'assemblage viral en induisant le multimérisation de Gag.Dans le travail présent, nous avons employé la méthode de purification d'affinité en tandem suivie de la spectrométrie de masse pour caractériser les RNPs contenus dans Staufen1. Nous nous sommes concentrés sur la recherche des protéines qui s'associent aux complexes Staufen1 isolés des cellules qui expriment ou non VIH-1. Ensuite, nous avons effectué une comparaison détaillée du contenu protéique entre Staufen1 sous forme native et Staufen1 complexé au RNPs VIH-1 et nous avons défini les modulations causées par l'expression de VIH-1.Nous avons utilisé les méthodes de complémentation par fluorescence bimoléculaire et trimoleculaire qui permettent la visualisation et la localisation directes des interactions protéine-protéine et des interactions protéine-protéine et d'ARN dans des cellules vivantes, pour prouver que Gag et Staufen1 interagissent comme démontré par la fluorescence ponctuée ou les vésicules dans les cellules. Nous avons démontré que les protéines partenaires s'associent principalement dans le cytoplasme. Cependant, nous avons également constaté qu'ils interagissent dans des microdomaines membranaires contenant du cholestérol enrichis en GM-1. De manière importante, Gag recrute spécifiquement Staufen1 au niveau de ces membranes de radeaux lipidiques, suggérant une fonction essentielle de ce facteur d'hôte pendant l'assemblage de Gag. En particulier, des expériences de TriFC dans lesquelles une protéine partenaire est attachée à l'ARNm ont permis de montrer des interactions Gag-Staufen1 indiquant un recrutement actif des protéines lorsqu'elles sont attachées à l'ARNm.De façon générale, les travaux présentent des recherches de protéomique fondamentale et de visualisation de cellules vivantes d'interaction virus-hôte. Ces expériences présentent la caractérisation complète de la composition de Staufen1 RNPs et démontre comment le VIH-1 s'adapte pour ses propres besoins. Cette thèse s'enrichit également de notre compréhension des mécanismes et des caractéristiques dynamiques spécifiques des interactions virus- hôte (Gag-Staufen1) dans des cellules vivantes, contrôlées par des techniques récentes de complémentation de fluorescence. Les résultats présentés ici sont considérables et contribuent à l'avancée des recherches dans le domaine du VIH-1, parce qu'une meilleure compréhension du mécanisme de l'ensemblage et du bourgeonnement du rétrovirus augmente la possibilité de développer de nouvelles thérapies antivirales, visant les étapes tardives critiques du cycle viral de réplication

Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1

Abstract Background Human immunodeficiency virus type 1 (HIV-1) uses cellular proteins and machinery to ensure transmission to uninfected cells. Although the host proteins involved in the transport of viral components toward the plasma membrane have been investigated, the dynamics of this process remain incompletely described. Previously we showed that the double-stranded (ds)RNA-binding protein, Staufen1 is found in the HIV-1 ribonucleoprotein (RNP) that contains the HIV-1 genomic RNA (vRNA), Gag and other host RNA-binding proteins in HIV-1-producing cells. Staufen1 interacts with the nucleocapsid domain (NC) domain of Gag and regulates Gag multimerization on membranes thereby modulating HIV-1 assembly. The formation of the HIV-1 RNP is dynamic and likely central to the fate of the vRNA during the late phase of the HIV-1 replication cycle. Results Detailed molecular imaging of both the intracellular trafficking of virus components and of virus-host protein complexes is critical to enhance our understanding of factors that contribute to HIV-1 pathogenesis. In this work, we visualized the interactions between Gag and host proteins using bimolecular and trimolecular fluorescence complementation (BiFC and TriFC) analyses. These methods allow for the direct visualization of the localization of protein-protein and protein-protein-RNA interactions in live cells. We identified where the virus-host interactions between Gag and Staufen1 and Gag and IMP1 (also known as VICKZ1, IGF2BP1 and ZBP1) occur in cells. These virus-host interactions were not only detected in the cytoplasm, but were also found at cholesterol-enriched GM1-containing lipid raft plasma membrane domains. Importantly, Gag specifically recruited Staufen1 to the detergent insoluble membranes supporting a key function for this host factor during virus assembly. Notably, the TriFC experiments showed that Gag and Staufen1 actively recruited protein partners when tethered to mRNA. Conclusions The present work characterizes the interaction sites of key components of the HIV-1 RNP (Gag, Staufen1 and IMP1), thereby bringing to light where HIV-1 recruits and co-opts RNA-binding proteins during virus assembly.</p

The TRAPP subunit Trs130p interacts with the GAP Gyp6p to mediate Ypt6p dynamics at the late Golgi

Small GTPases of the Rab superfamily participate in virtually all vesicle-mediated trafficking events. Cycling between an active GTP-bound form and an inactive GDP-bound form is accomplished in conjunction with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Rab cascades have been described in which an effector of an activated Rab is a GEF for a downstream Rab, thus ensuring activation of a pathway in an ordered fashion. Much less is known concerning crosstalk between GEFs and GAPs although regulation between these factors could also contribute to the overall physiology of a cell. Here we demonstrate that a subunit of the TRAPP II multisubunit tethering factor, a GEF for the GTPase Ypt1p, participates in the recruitment of Gyp6p, a GAP for the GTPase Ypt6p, to Golgi membranes. The extreme carboxy-terminal portion of the TRAPP II subunit Trs130p is required for the interaction between TRAPP II and Gyp6p. We further demonstrate that TRAPP II mutants, but not a TRAPP III mutant, display a defect in Gyp6p interaction. A consequence of this defective interaction is the enhanced localization of Ypt6p at late Golgi membranes. Although a ypt31/32 mutant also resulted in an enhanced localization of Gyp6p at the late Golgi, the effect was not as dramatic as that seen for TRAPP II mutants, nor was Ypt31/32 detected in the same TRAPP II purification that detected Gyp6p. We propose that the interaction between TRAPP II and Gyp6p represents a parallel mechanism in addition to that mediated by Ypt31/32 for the recruitment of a GAP to the appropriate membrane, and is a novel example of crosstalk between a Rab GAP and GEF

Characterization of Staufen1 ribonucleoproteins by mass spectrometry and biochemical analyses reveal the presence of diverse host proteins associated with human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) unspliced, 9kb genomic RNA (vRNA) is exported from the nucleus for the synthesis of viral structural proteins and enzymes (Gag and Gag/Pol) and is then transported to sites of virus assembly where it is packaged into progeny virions. vRNA co-exists in the cytoplasm in the context of the HIV-1 ribonucleoprotein (RNP) that is currently defined by the presence of Gag and several host proteins including the double-stranded RNA-binding protein, Staufen1. In this study we isolated Staufen1 RNP complexes derived from HIV-1-expressing cells using tandem affinity purification and have identified multiple host protein components by mass spectrometry. Four viral proteins, including Gag, Gag/Pol, Env and Nef as well as &gt;200 host proteins were identified in these RNPs. Moreover, HIV-1 induces both qualitative and quantitative differences in host protein content in these RNPs. 22% of Staufen1-associated factors are virion-associated suggesting that the RNP could be a vehicle to achieve this. In addition, we provide evidence on how HIV-1 modulates the composition of cytoplasmic Staufen1 RNPs. Biochemical fractionation by density gradient analyses revealed new facets on the assembly of Staufen1 RNPs. The assembly of dense Staufen1 RNPs that contain Gag and several host proteins were found to be entirely RNA-dependent but their assembly appeared to be independent of Gag expression. Gag-containing complexes fractionated into a lighter and another, more dense pool. Lastly, Staufen1 depletion studies demonstrated that the previously characterized Staufen1 HIV-1-dependent RNPs are most likely aggregates of smaller RNPs that accumulate at juxtanuclear domains. The molecular characterization of Staufen1 HIV-1 RNPs will offer important information on virus-host cell interactions and on the elucidation of the function of these RNPs for the transport of Gag and the fate of the unspliced, vRNA in HIV-1-producing cells

Giant primary retroperitoneal seminoma: A case report

Introduction. Primary extragonadal seminomas are rare tumors. There have been only a few cases of the primary retroperitoneal seminomas reported in the literature up to date. Case report. We reported a 56-year-old man with giant primary retroperitoneal seminoma presented with the enlargement of the left side of the abdomen and deep venous thrombosis of the left leg. Computed tomography of the abdomen showed a large tumor occupying the left part of the retroperitoneal space with 23 x 13 cm in diameter. Firm tumor mass having 25 x 15 cm in diameter was surgically removed from the left retroperitoneum. The tumor adhered the tunica adventitia of the aorta and it was carefully resected from the aortic wall. The diagnosis of seminoma was made during histopathological examination. The patient underwent chemotherapy. Two years after finished chemotherapy the patient accepted left orchiectomy with the aim of eliminating the possibility of the occult malignancy of the testicle. Histopathological analysis of the testicular tissue was normal and the diagnosis of primary retroperitoneal seminoma was confirmed. Conclusion. Despite its small incidence in general population, the diagnosis of retroperitoneal seminoma should be considered in male patients with nonspecific symptoms and with retroperitoneal tumor mass