Hepatitis C virus is released via a non-canonical secretory route.

We analyzed HCV morphogenesis using viral genomes encoding for a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization and replication kinetics were comparable to untagged HCV and E1-mCherry tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and non-structural NS5A followed a tempo-spatial pattern with succinct decrease in replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating absence of viral glycoprotein processing by the Golgi. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggests that assembled HCV particles are released via a non-canonical secretory route involving the endosomal compartment. IMPORTANCE STATEMENT: The goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which result in the production of fluorescently labeled viral particles. We used live cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap with Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point towards the possible existence of a not yet described cellular secretion route

The Rabies Virus Interferon Antagonist P Protein Interacts with Activated STAT3 and Inhibits Gp130 Receptor Signaling

Fulltext embargoed for: 6 months post date of publicationImmune evasion by rabies virus depends on targeting of the signal transducers and activator of transcription 1 (STAT1) and STAT2 proteins by the viral interferon antagonist P protein, but targeting of other STAT proteins has not been investigated. Here, we find that P protein associates with activated STAT3 and inhibits STAT3 nuclear accumulation and Gp130-dependent signaling. This is the first report of STAT3 targeting by the interferon antagonist of a virus other than a paramyxovirus, indicating that STAT3 antagonism is important to a range of human-pathogenic viruses

AP-2 is the crucial clathrin adaptor protein for CD4 downmodulation by HIV-1 Nef in primary infected CD4+ T cells.

HIV-1 Nef-mediated CD4 downmodulation involves various host factors. We investigated the importance of AP-1, AP-2, AP-3, V1H-ATPase, β-COP and ACOT8 for CD4 downmodulation in HIV-1 infected shRNA-expressing CD4+ T cells and characterized direct interaction with Nef by FRET. Binding of lentiviral Nefs to CD4 and AP-2 was conserved and only AP-2 knockdown impaired Nef-mediated CD4 downmodulation from primary T cells. Altogether, among the factors tested, AP-2 is the most important player for Nef-mediated CD4 downmodulation

PARAMOUNT LEVELS OF ERGOTHIONEINE TRANSPORTER SLC22A4 MRNA IN BOAR SEMINAL VESICLES AND CROSS-SPECIES ANALYSIS OF ERGOTHIONEINE AND GLUTATHIONE IN SEMINAL PLASMA

INTRODUCTION Ergothioneine (ET) is a natural antioxidant which is biosynthesized solely by fungi and mycobacteria (1). Chemically, ET is the betaine of histidine with a sulfur atom attached to the imidazole ring. It should not be considered a thiol compound, but rather a thione, a derivative of thiourea. As a consequence of the prevailing thione tautomer, ET is a very stable antioxidant with unique properties (2). Recently, we have discovered an ET transporter (ETT; gene symbol SLC22A4) (3). ETT from human (ETTh) has high affinity for ET (K m =21 µmol/l) and catalyzes cotransport of ET with Na + . Cells lacking ETT do not accumulate ET, since the plasma membrane is virtually impermeable for this compound. By contrast, cells with expression of ETT accumulate ET to high levels. In humans, ETT is strongly expressed in small intestine, kidney, erythrocyte progenitor cells in bone marrow, and monocytes (3). Much interest in ETT has been generated by casecontrol studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases such as Crohn's disease (4-9), ulcerative colitis (10) and Type I diabetes (11). It is presently unknown how the mutation in the transporter gene promotes disease. The existence of a specific transporter suggests a beneficial role for ET. Most authors consider ET as an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. A particularly conspicuous site of ET accumulation is boar semen; in seminal plasma, 0.3-0.8 mmol/l ET has been measured (12, 13). Boar spermatozoa contain, if any at all, much less ET than seminal plasma (14). Thus, in boar semen ET is an extracellular constituent -unlike blood, where nearly all ET is contained within erythrocytes and specific leukocytes. ET apparently enters boar semen at the seminal vesicles. The isolated secretion of this gland contains amazingly high concentrations (average: 3.4 mmol/l; range: 1.3-11 mM; 29-256 mg/100 ml) (12); this level is 10 times higher than in the blood of pig (range: 0.13-1.2 mM; 3-27 mg/100 ml) or other specie

Conservation of a unique mechanism of immune evasion across the Lyssavirus genus.

International audienceThe evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses

Interaction of Rabies Virus P-Protein With STAT Proteins is Critical to Lethal Rabies Disease

BACKGROUND: Rabies virus (RABV) causes rabies disease resulting in >55,000 human deaths/year. The multifunctional RABV P-protein has essential roles in genome replication, and forms interactions with cellular STAT proteins that are thought to underlie viral antagonism of interferon-dependent immunity. However, the molecular details of P-protein-STAT interaction, and its importance to disease are unresolved. METHODS: Studies were performed using sequence/structure analysis, mutagenesis, immunoprecipitation, luciferase and qRT-PCR-based signaling assays, confocal microscopy and reverse genetics/in vivo infection. RESULTS: We identified a hydrophobic pocket of the P-protein C-terminal domain as critical to STAT-binding/antagonism. This interface was found to be functionally and spatially independent of the region responsible for N-protein interaction, which is critical to genome replication. Based on these findings, we generated the first mutant RABV lacking STAT-association. Growth of the virus in vitro was unimpaired, but it lacked STAT-antagonist function and was highly sensitive to interferon. Importantly, growth of the virus was strongly attenuated in brains of infected mice, producing no major neurological symptoms, compared with the invariably lethal wild-type virus. CONCLUSIONS: These data represent direct evidence that P-protein-STAT interaction is critical to rabies, and provide novel insights into the mechanism by which RABV coordinates distinct functions in interferon antagonism and replication