A 3D organoid platform that supports liver-stage P.falciparum infection can be used to identify intrahepatic antimalarial drugs

Malaria, a major public health burden, is caused by Plasmodium spp parasites that first replicate in the human liver to establish infection before spreading to erythrocytes. Liver-stage malaria research has remained challenging due to the lack of a clinically relevant and scalable in vitro model of the human liver. Here, we demonstrate that organoids derived from intrahepatic ductal cells differentiated into a hepatocyte-like fate can support the infection and intrahepatic maturation of Plasmodium falciparum. The P.falciparum exoerythrocytic forms observed expressed both early and late-stage parasitic proteins and decreased in frequency in response to treatment with both known and putative antimalarial drugs that target intrahepatic P.falciparum. The P.falciparum-infected human liver organoids thus provide a platform not only for fundamental studies that characterise intrahepatic parasite-host interaction but can also serve as a powerful translational tool in pre-erythrocytic vaccine development and to identify new antimalarial drugs that target the liver stage infection.</p

A 3D organoid platform that supports liver-stage P.falciparum infection can be used to identify intrahepatic antimalarial drugs

Malaria, a major public health burden, is caused by Plasmodium spp parasites that first replicate in the human liver to establish infection before spreading to erythrocytes. Liver-stage malaria research has remained challenging due to the lack of a clinically relevant and scalable in vitro model of the human liver. Here, we demonstrate that organoids derived from intrahepatic ductal cells differentiated into a hepatocyte-like fate can support the infection and intrahepatic maturation of Plasmodium falciparum. The P.falciparum exoerythrocytic forms observed expressed both early and late-stage parasitic proteins and decreased in frequency in response to treatment with both known and putative antimalarial drugs that target intrahepatic P.falciparum. The P.falciparum-infected human liver organoids thus provide a platform not only for fundamental studies that characterise intrahepatic parasite-host interaction but can also serve as a powerful translational tool in pre-erythrocytic vaccine development and to identify new antimalarial drugs that target the liver stage infection.</p

Ebola virus VP35 blocks stress granule assembly

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

PCID2 dysregulates transcription and viral RNA processing to promote HIV-1 latency

HIV-1 latency results from tightly regulated molecular processes that act at distinct steps of HIV-1 gene expression. Here, we characterize PCI domain-containing 2 (PCID2) protein, a subunit of the transcription and export complex 2 (TREX2) complex, to enforce transcriptional repression and post-transcriptional blocks to HIV-1 gene expression during latency. PCID2 bound the latent HIV-1 LTR (long terminal repeat) and repressed transcription initiation during latency. Depletion of PCID2 remodeled the chromatin landscape at the HIV-1 promoter and resulted in transcriptional activation and latency reversal. Immunoprecipitation coupled to mass spectrometry identified PCID2-interacting proteins to include negative viral RNA (vRNA) splicing regulators, and PCID2 depletion resulted in over-splicing of intron-containing vRNA in cell lines and primary cells obtained from PWH. MCM3AP and DSS1, two other RNA-binding TREX2 complex subunits, also inhibit transcription initiation and vRNA alternative splicing during latency. Thus, PCID2 is a novel HIV-1 latency-promoting factor, which in context of the TREX2 sub-complex PCID2-DSS1-MCM3AP blocks transcription and dysregulates vRNA processing.</p

Catchet-MS identifies IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5′LTR. Catchet-MS identified known and novel latent 5′LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs. We demonstrate that, by targeting IKZF1 for degradation, these compounds reverse HIV-1 latency in CD4+ T-cells isolated from virally suppressed people living with HIV-1 and that they are able to synergize with other known LRAs

The BAF complex inhibitor pyrimethamine reverses HIV-1 latency in people with HIV-1 on antiretroviral therapy

Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.</p

Sex differences in HIV-1 persistence and the implications for a cure

Of the 38 million people currently living with Human Immunodeficiency Virus type-1 (HIV-1), women, especially adolescents and young women, are disproportionally affected by the HIV-1 pandemic. Acquired immunodeficiency syndrome (AIDS) - related illnesses are the leading cause of death in women of reproductive age worldwide. Although combination antiretroviral therapy (cART) can suppress viral replication, cART is not curative due to the presence of a long-lived viral reservoir that persists despite treatment. Biological sex influences the characteristics of the viral reservoir as well as the immune responses to infection, factors that can have a significant impact on the design and quantification of HIV-1 curative interventions in which women are grossly underrepresented. This mini-review will provide an update on the current understanding of the impact of biological sex on the viral reservoir and will discuss the implications of these differences in the context of the development of potential HIV-1 curative strategies, with a focus on the shock and kill approach to an HIV-1 cure. This mini-review will also highlight the current gaps in the knowledge of sex-based differences in HIV-1 persistence and will speculate on approaches to address them to promote the development of more scalable, effective curative approaches for people living with HIV-1

The effect of host mRNA decay proteins on HIV-1 genomic RNA metabolism and viral gene expression

mRNA surveillance pathways are host quality control mechanisms that degrade aberrant mRNA to prevent the accumulation of potentially toxic truncated or misfolded proteins. Substrates for mRNA surveillance include transcripts that have pre-termination codons, long 3’ untranslated regions (UTRs), retained introns or upstream open reading frames. The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) also contains long 3’ UTRs. However, not only is the vRNA able to evade mRNA surveillance, HIV-1 has also been demonstrated to recruit the mRNA decay proteins to promote vRNA stability and ensure viral gene expression. UPF1 is an integral protein of the nonsense-mediated mRNA decay pathway and Staufen1 is also involved in the post-transcriptional events of mRNA decay, mRNA trafficking and translation. In this work, we further elucidated the roles of these host mRNA decay proteins on the post-transcriptional regulation of HIV-1 vRNA metabolism. The ability of HIV-1 to form a stable viral reservoir is the major obstacle to an HIV-1 cure. In our studies investigating HIV-1 latency, we characterised the roles of mRNA decay proteins on the maintenance of viral latency in a latently-infected model T cell line using fluorescence in situ hybridisation - Flow Cytometry (FISH-flow). We observed that UPF1 enhances vRNA stability, thus promoting viral gene expression at a post-transcriptional level. We also demonstrated that two other proteins involved in nonsense-mediated mRNA decay, UPF2 and SMG6, are negative regulators of proviral reactivation and reduce viral gene expression in the same model T cell line. In primary HIV-1-infected CD4+ T cells, UPF1 also enhanced vRNA stability and viral gene expression. UPF2 and SMG6 were also found to restrict HIV-1 gene expression in primary monocyte-derived macrophages, another viral reservoir of HIV-1 infection. In related studies investigating the host response to viral infection, we characterised a novel role for Staufen1 in the rescue of cellular mRNA translation and viral gene expression during HIV-1 nucleocapsid (NC)-induced stress granule assembly. Altogether, the host mRNA decay proteins UPF1, UPF2, SMG6 and Staufen1 affect various stages of vRNA metabolism and HIV-1 replication. These findings can be applied towards an HIV-1 cure using two strategies: the ‘kick and kill’ strategy to bolster the reactivation of the HIV-1 provirus and effectively decrease the size of the viral reservoir, or a ‘block and lock’ strategy to permanently silence the HIV-1 provirus.Les mécanismes de surveillance de l’ARN messager (ARNm) sont des voies cellulaires de control de qualité permettant de dégrader les ARNm défectueux afin d’éviter l’accumulation de protéines tronquées ou mal repliées qui seraient potentiellement toxiques pour la cellule. Les processus de surveillance de l’ARNm ciblent les transcrits contenant des codons pré-terminaux, de longues régions non traduites (unstranslated regions ; UTR), des introns retenus, ou des cadres de lectures ouverts situés en amont de la séquence codante. L’ARN génomique viral (ARNv) du virus de l’immunodéficience humaine (VIH) contient de longues régions 3’-UTRs. Cependant, non seulement l’ARNv est capable d’échapper à la surveillance cellulaire de l’ARNm, mais le VIH détourne également les protéines de dégradation de l’ARNm afin d’assurer la stabilité de son propre ARNv et l’expression de ses gènes. UPF1 est une protéine essentielle du mécanisme de dégradation de l’ARNm non-sens ; Staufen1 est également impliquée dans les évènements post-transcriptionnels de dégradation de l’ARNm, de circulation de l’ARNm et de translation. Cette étude clarifie les rôles de ces protéines cellulaires de dégradation de l’ARNm dans la régulation post-transcriptionnelle du métabolisme de l’ARNv du VIH-1. L’aptitude du VIH à former un réservoir viral stable est un obstacle majeur dans le développement d’un remède contre le VIH. Nos travaux ont permis de définir le rôle des protéines de dégradation de l’ARNm dans le maintien de la latence virale grâce à l’étude par hybridation in situ en fluorescence – cytométrie en flux (FISH-Flow) d’un modèle de cellules T infectées par la forme latente du virus. Nous avons démontré que UPF1 améliore la stabilité de l’ARNv, favorisant ainsi l’expression des gènes viraux au niveau post-transcriptionnel. Nous avons également prouvé dans ce même modèle cellulaire que deux autres protéines, UPF2 et SMG6, impliquées dans la dégradation de l’ARNm non-sens, régulent négativement la réactivation du provirus et diminuent l’expression des gènes viraux. Dans le cas de lymphocytes T CD4+ primaires infectés par le VIH, UPF1 augmente la stabilité de l’ARNm et l’expression des gènes viraux. UPF2 et SMG6 restreignent également l’expression des gènes du VIH dans des macrophages primaires dérivés de monocytes, d’autres cellules réservoirs de l’infection au VIH. Dans une étude connexe sur la réponse cellulaire à une infection virale, nous avons caractérisé un nouveau rôle de Staufen 1 dans le rétablissement de la traduction de l’ARNm cellulaire et dans l’expression des gènes viraux lors de l’assemblage de granules de stress induit par les protéines nucléocapside (NC) du VIH-1. En conclusion, les protéines cellulaires de dégradation de l’ARNm - UPF1, UPF2, SMG6 et Staufen1 - affectent divers stages du métabolisme de l’ARNv et de la réplication du VIH-1. Ces découvertes peuvent être appliquées au développement d’un remède contre le VIH par le biais de deux stratégies : la stratégie « kick and kill » pour stimuler la réactivation du provirus et diminuer efficacement le réservoir viral ; ou la stratégie « block and lock » pour bloquer de façon permanente la réactivation du virus et le garder sous sa forme latente

DEAD-ly Affairs: The Roles of DEAD-Box Proteins on HIV-1 Viral RNA Metabolism

In order to ensure viral gene expression, Human Immunodeficiency virus type-1 (HIV-1) recruits numerous host proteins that promote optimal RNA metabolism of the HIV-1 viral RNAs (vRNAs), such as the proteins of the DEAD-box family. The DEAD-box family of RNA helicases regulates multiple steps of RNA metabolism and processing, including transcription, splicing, nucleocytoplasmic export, trafficking, translation and turnover, mediated by their ATP-dependent RNA unwinding ability. In this review, we provide an overview of the functions and role of all DEAD-box family protein members thus far described to influence various aspects of HIV-1 vRNA metabolism. We describe the molecular mechanisms by which HIV-1 hijacks these host proteins to promote its gene expression and we discuss the implications of these interactions during viral infection, their possible roles in the maintenance of viral latency and in inducing cell death. We also speculate on the emerging potential of pharmacological inhibitors of DEAD-box proteins as novel therapeutics to control the HIV-1 pandemic