Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified cellular serine/threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)-mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA ). In addition, a humanized liver Fah-/- /Rag2-/- /Il2rg-/- (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. CONCLUSION: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV-mediated carcinogenesis. (Hepatology 2017;66:1750-1765).

Selective acquisition of host-derived ICAM-1 by HIV-1 is a matrix-dependent process.

UNLABELLED: HIV-1 acquires an impressive number of foreign components during its formation. Despite all previous efforts spent studying the nature and functionality of virus-anchored host molecules, the exact mechanism(s) through which such constituents are acquired by HIV-1 is still unknown. However, in the case of ICAM-1, one of the most extensively studied transmembrane proteins found associated with mature virions, the Pr55(Gag) precursor polyprotein appears to be a potential interaction partner. We investigated and characterized at the molecular level the process of ICAM-1 incorporation using initially a Pr55(Gag)-based virus-like particle (VLP) model. Substitution of various domains of Pr55(Gag), such as the nucleocapsid, SP2, or p6, had no effect on the acquisition of ICAM-1. We found that the structural matrix protein (MA) is mandatory for ICAM-1 incorporation within VLPs, and we confirmed this novel observation with the replication-competent HIV-1 molecular clone NL4.3. Additional studies suggest that the C-terminal two-thirds of MA, and especially 13 amino acids positioned inside the fifth α-helix, are important. Moreover, based on three-dimensional (3D) modeling of protein-protein interactions (i.e., protein-protein docking) and further validation by a virus capture assay, we found that a series of acidic residues in the MA domain interact with basic amino acids located in the ICAM-1 cytoplasmic tail. Our findings provide new insight into the molecular mechanism governing the acquisition of ICAM-1, a host molecule known to enhance HIV-1 infectivity in a significant manner. Altogether, these observations offer a new avenue for the development of antiviral therapeutics that are directed at a target of host origin. IMPORTANCE: Intercellular adhesion molecule 1 (ICAM-1) is a cell surface host component known to be efficiently inserted within emerging HIV-1 particles. It has been demonstrated that host-derived ICAM-1 molecules act as a strong attachment factor and increase HIV-1 infectivity substantially. Despite previous efforts spent studying virus-associated host molecules, the precise mechanism(s) through which such constituents are inserted within emerging HIV-1 particles still remains obscure. Previous data suggest that the Pr55(Gag) precursor polyprotein appears as a potential interaction partner with ICAM-1. In the present study, we demonstrate that the HIV-1 matrix domain plays a key role in the ICAM-1 incorporation process. Some observations were confirmed with whole-virus preparations amplified in primary human cells, thereby providing physiological significance to our data.

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes.

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV-1 co-infections. We have delineated the mechanism of cell death induced by the HIV-1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir-Leishmania interactions, we selected Nelfinavir-resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir-resistant and -sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir-resistant and -sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time-dependent manner. Furthermore, high-resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir-resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug-induced intracellular vesicles.

Exposure of human astrocytes to leukotriene C4 promotes a CX3CL1/fractalkine-mediated transmigration of HIV-1-infected CD4⁺ T cells across an in vitro blood-brain barrier model.

Eicosanoids, including cysteinylleukotrienes (cysLTs), are found in the central nervous system (CNS) of individuals infected with HIV-1. Few studies have addressed the contribution of cysLTs in HIV-1-associated CNS disorders. We demonstrate that conditioned medium from human astrocytes treated with leukotriene C4 (LTC4) increases the transmigration of HIV-1-infected CD4(+) T cells across an in vitro blood-brain barrier (BBB) model using cultured brain endothelial cells. Additional studies indicate that the higher cell migration is linked with secretion by astrocytes of CX3CL1/fractalkine, a chemokine that has chemoattractant activity for CD4(+) T cells. Moreover, we report that the enhanced cell migration across BBB leads to a more important CD4(+) T cell-mediated HIV-1 transfer toward macrophages. Altogether data presented in the present study reveal the important role that LTC4, a metabolite of arachidonic acid, may play in the HIV-1-induced neuroinvasion, neuropathogenesis and disease progression.

Efficient production of HIV-1 virus-like particles from a mammalian expression vector requires the N-terminal capsid domain.

It is now well accepted that the structural protein Pr55(Gag) is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55(Gag) is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55(Gag) sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55(Gag)-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development.