Project description:Hepatoviruses, a common cause of acute hepatitis in humans, are atypical picornaviruses released from cells in small vesicles resembling exosomes. We show the nonlytic release of these quasi-enveloped virions is mediated by a C-terminal extension of the VP1 capsid protein (pX) that binds the Bro1 domains of ALIX and HD-PTP, and recruits the ubiquitin ligase ITCH to drive an association with endosomal sorting complexes required for transport (ESCRT). Fusing pX to a self-assembling, de novo designed nanocage protein resulted in ESCRT-dependent release mediated by a 20-amino acid core sequence containing a Y[KR]xxR[LM] motif conserved in viruses from bats to humans. Mutations in this motif ablate release and lead virus to accumulate intracellularly. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies, identifies its key cellular binding partners, and shows non-lytic release of quasi-enveloped virus is a tightly orchestrated and ancient evolutionary trait of hepatoviruses.
Project description:The overexpression and misfolding of viral proteins in the endoplasmic reticulum (ER) may cause cellular stress, thereby inducing cytoprotective proteostatic host responses involving phosphorylation of eIFa. Here, we show the positive-strand RNA virus responsible for infectious hepatitis adopts a stress-resistant, phospho-eIF2a independent mechanism of translation that ensures synthesis of its viral proteins within the infected liver. Cap-independent hepatovirus translation and productive infection in vivo requires PDAP1, a small phosphoprotein with previously unrecognized eIF4E-binding activity. We show PDAP1 interacts also with eIF1A, and is essential for translation of stress-resistant host mRNAs that evade the proteostatic response to ER stress.
Project description:Hepatitis C Virus protein NS5A was found to upregulate assembly of cap binding initiation complex eIF4F in Huh7.5 cells. NS5A also was found to associate with translation machinery. To understand consequences of NS5A mediation in host translation, we analyzed mRNA associated with polysome fractions of NS5A expressing Huh7.5 cells and compared them with the corresponding fractions from control cells. Agilent-027114 Genotypic Technology designed Custom Human Whole Genome 8x60k Microarray
Project description:A knockout cell library in Huh7.5.1 cells was generated by introducing a genome-scale CRISPR library (GeCKOv2, Addgene #1000000049) and subjected to hepatitis A virus infection (HM175/18f) to isolate virus-resistant mutant cells. Genomic DNA was isolated from the original and virus-selected mutant cell populations and abundance of guideRNA encoding sequences were measured by sequencing on an Illumina NextSeq (High Output).
