Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.

Highlights d Retroviral Gag proteins have conserved intrinsically disordered prion-like domains d Pan-retrovirus family nucleocapsid proteins induce liquidliquid phase separation d Nucleocapsid protein phase separation and stress granule assembly is Zn 2+ dependent d Zn 2+ chelators and ejectors induce nuclear repositioning of the genomic RNA

The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, HBeAg, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV infected patients.

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNβ. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.

Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly.

BackgroundThe ability of human immunodeficiency virus type 1 (HIV-1) to form a stable viral reservoir is the major obstacle to an HIV-1 cure and post-transcriptional events contribute to the maintenance of viral latency. RNA surveillance proteins such as UPF1, UPF2 and SMG6 affect RNA stability and metabolism. In our previous work, we demonstrated that UPF1 stabilises HIV-1 genomic RNA (vRNA) and enhances its translatability in the cytoplasm. Thus, in this work we evaluated the influence of RNA surveillance proteins on vRNA expression and, as a consequence, viral reactivation in cells of the lymphoid lineage.MethodsQuantitative fluorescence in situ hybridisation—flow cytometry (FISH-flow), si/shRNA-mediated depletions and Western blotting were used to characterise the roles of RNA surveillance proteins on HIV-1 reactivation in a latently infected model T cell line and primary CD4+ T cells.ResultsUPF1 was found to be a positive regulator of viral reactivation, with a depletion of UPF1 resulting in impaired vRNA expression and viral reactivation. UPF1 overexpression also modestly enhanced vRNA expression and its ATPase activity and N-terminal domain were necessary for this effect. UPF2 and SMG6 were found to negatively influence viral reactivation, both via an interaction with UPF1. UPF1 knockdown also resulted in reduced vRNA levels and viral gene expression in HIV-1-infected primary CD4+ T cells.ConclusionOverall, these data suggest that RNA surveillance proteins affect HIV-1 gene expression at a post-transcriptional level. An elucidation of the role of vRNA metabolism on the maintenance of HIV-1 persistence can lead to the development of novel curative strategies.Electronic supplementary materialThe online version of this article (10.1186/s12977-018-0425-2) contains supplementary material, which is available to authorized users.

The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) has two major fates during viral replication: to serve as the template for the major structural and enzymatic proteins, or to be encapsidated and packaged into assembling virions to serve as the genomic vRNA in budding viruses. The dynamic balance between vRNA translation and encapsidation is mediated by numerous host proteins, including Staufen1. During HIV-1 infection, HIV-1 recruits Staufen1 to assemble a distinct ribonucleoprotein complex promoting vRNA encapsidation and viral assembly. Staufen1 also rescues vRNA translation and gene expression during conditions of cellular stress. In this work, we utilized novel Staufen1 −/− gene-edited cells to further characterize the contribution of Staufen1 in HIV-1 replication. We observed a marked deficiency in the ability of HIV-1 to dissociate stress granules (SGs) in Staufen1-deficient cells and remarkably, the vRNA repositioned to SGs. These phenotypes were rescued by Staufen1 expression in trans or in cis, but not by a dsRBD-binding mutant, Staufen1F135A. The mistrafficking of the vRNA in these Staufen1 −/− cells was also accompanied by a dramatic decrease in viral production and infectivity. This work provides novel insight into the mechanisms by which HIV-1 uses Staufen1 to ensure optimal vRNA translation and trafficking, supporting an integral role for Staufen1 in the HIV-1 life cycle, positioning it as an attractive target for next-generation antiretroviral agents.

The nucleocapsid (NC) is an N-terminal protein derived from the HIV-1 Gag precursor polyprotein, pr55 NC possesses key functions at several pivotal stages of viral replication. For example, an interaction between NC and the host double-stranded RNA-binding protein Staufen1 was shown to regulate several steps in the viral replication cycle, such as Gag multimerization and genomic RNA encapsidation. In this work, we observed that the overexpression of NC leads to the induction of stress granule (SG) assembly. NC-mediated SG assembly was unique as it was resistant to the SG blockade imposed by the HIV-1 capsid (CA), as shown in earlier work. NC also reduced host cell mRNA translation, as judged by a puromycylation assay of de novo synthesized proteins, and this was recapitulated in polysome profile analyses. Virus production was also found to be significantly reduced. Finally, Staufen1 expression completely rescued the blockade to NC-mediated SG assembly, global mRNA translation as well as virus production. NC expression also resulted in the phosphorylation of protein kinase R (PKR) and eIF2α, and this was inhibited with Staufen1 coexpression. This work sheds light on an unexpected function of NC in host cell translation. A comprehensive understanding of the molecular mechanisms by which a fine balance of the HIV-1 structural proteins NC and CA act in concert with host proteins such as Staufen1 to modulate the host stress response will aid in the development of new antiviral therapeutics.