The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids.

BACKGROUND & AIMS: Hepatitis B virus (HBV) has a DNA genome but replicates within the nucleus by reverse transcription of an RNA pregenome, which is converted to DNA in cytoplasmic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are a major target for T cell-mediated immune responses. We investigated the mechanism of cytoplasmic capsid transport, which is important for infection but also for cytosolic capsid removal. METHODS: We used virion-derived capsids containing mature rcDNA (matC) and empty capsids (empC). RNA-containing capsids (rnaC) were used as a control. The investigations comprised pull-down assays for identification of cellular interaction partners, immune fluorescence microscopy for their colocalization and electron microscopy after microinjection to determine their biological significance. RESULTS: matC and empC underwent active transport through the cytoplasm towards the nucleus, while rnaC was poorly transported. We identified the dynein light chain LL1 as a functional interaction partner linking capsids to the dynein motor complex and showed that there is no compensatory transport pathway. Using capsid and dynein LL1 mutants we characterized the required domains on the capsid and LL1. CONCLUSIONS: This is the first investigation on the detailed molecular mechanism of how matC pass the cytoplasm upon infection and how empC can be actively removed from the cytoplasm into the nucleus. Considering that hepatocytes with cytoplasmic capsids are better recognized by the T cells, we hypothesize that targeting capsid DynLL1-interaction will not only block HBV infection but also stimulate elimination of infected cells. LAY SUMMARY: In this study, we identified the molecular details of HBV translocation through the cytoplasm. Our evidence offers a new drug target which could not only inhibit infection but also stimulate immune clearance of HBV infected cells.

TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the flagellar pocket collar (FPC). TbBILBO1 is the first-described FPC protein of Trypanosoma brucei. It is essential for parasite survival, FP and FPC biogenesis. In this work, we characterize TbKINX1B, a novel TbBILBO1 partner. We demonstrate that TbKINX1B is located on the basal bodies, the microtubule quartet (a set of four microtubules) and the FPC in T. brucei. Down-regulation of TbKINX1B by RNA interference in bloodstream forms is lethal, inducing an overall disturbance in the endomembrane network. In procyclic forms, the RNAi knockdown of TbKINX1B leads to a minor phenotype with a small number of cells displaying epimastigote-like morphologies, with a misplaced kinetoplast. Our results characterize TbKINX1B as the first putative kinesin to be localized both at the basal bodies and the FPC with a potential role in transporting cargo along with the microtubule quartet.

Title: TbKINX1B, un nouveau partenaire de BILBO1, et une protéine essentielle dans la forme sanguine de Trypanosoma brucei. Abstract: La poche flagellaire (PF) de l'agent pathogène Trypanosoma brucei est une structure importante à copie unique formée par l'invagination de la membrane pelliculaire. Elle est le site unique de l'endo- et de l'exocytose et est nécessaire à la pathogénicité du parasite. La PF est constituée de sous-domaines structurels distincts, le moins exploré étant le collier de poche flagellaire (CPF). TbBILBO1 est la première protéine du CPF décrite. Elle est essentielle pour la survie du parasite et la biogenèse de la PF et du CPF. Dans ce travail, nous caractérisons TbKINX1B, un nouveau partenaire de TbBILBO1. Nous démontrons que TbKINX1B est localisée au niveau des corps basaux, du quartet de microtubules (un ensemble de quatre microtubules) et du CPF chez T. brucei. La diminution de l'expression de TbKINX1B par ARN interférence dans les formes sanguines est létale, induisant une perturbation globale du réseau endomembranaire. Dans les formes procycliques, l'ARN interférence conduit à un phénotype mineur avec un petit nombre de cellules présentant des morphologies de type épimastigote, avec un kinétoplaste mal placé. Nos résultats caractérisent TbKINX1B comme la première kinésine putative à être localisée à la fois au niveau des corps basaux et du CPF avec un rôle potentiel dans le transport de cargaison le long du quartet de microtubules.