Human LC-MS (Ubiquitin Ligase ITCH Controls SARS-CoV-2 Virion Assembly and Egress)
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Qiwang Xiang
LAB HEAD: Qiwang Xiang
PROVIDER: PXD049221 | Pride | 2026-02-23
REPOSITORIES: Pride
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Project description:To identify host proteins that interact with ITCH, we performed proteomic analysis of the co-immunoprecipitates pulled down by the ITCH expressed in HEK293 cells.
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Project description:Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from four experiments with samples obtained in four independent RNA immunoprecipitations identified 375 transcripts that co-immunoprecipitate with FLAG RHA. Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs HEK293 cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe a human Agilent expression arrays. An immunoprecipitation with cells transfected with empty FLAG plasmid was used as negative control.
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Project description:Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from duplicate experiments with samples obtained in two independent RNA immunoprecipitations identified 407 transcripts that co-immunoprecipitate with FLAG RHA. Keywords: gene expression array-based Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs COS cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were pooled and immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe rhesus macaque Affimetrix expression arrays. Steady state mRNA levels were monitored on a separate array probed with total RNA.
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Project description:The human cytomegalovirus virion is composed of a DNA genome packaged in an icosahedral capsid, surrounded by a tegument of protein and RNA, all enclosed within a glycoprotein-studded envelope. Achieving this intricate virion architecture requires a coordinated process of assembly and egress. We show here that pUL71, a component of the virion tegument with a previously uncharacterized function, is required for the virus-induced reorganization of host cell membranes, which is necessary for efficient viral assembly and egress. A mutant that did not express pUL71 was able to efficiently accumulate viral genomes and proteins that were tested but was defective for the production and release of infectious virions. The protein localized to vesicular structures at the periphery of the viral assembly compartment, and during infection with a pUL71-deficient virus, these structures were grossly enlarged and aberrantly contained a cellular marker of late endosomes/lysosomes. Mutant virus preparations exhibited less infectivity per unit genome than wild-type virus preparations, due to aggregation of virus particles and their association with membrane fragments. Finally, mutant virus particles accumulated within the cytoplasm of infected cells and were localized to the periphery of large structures with properties of lysosomes, whose formation was kinetically favored in mutant-virus-infected cells. Together, these observations point to a role for pUL71 in the establishment and/or maintenance of a functional viral assembly compartment that is required for normal virion trafficking and egress from infected cells.
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Project description:Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gDΔct) and the entire gE gene (ΔgE) did not exhibit substantial defects in cytoplasmic virion envelopment and egress (H. C. Lee et al., J. Virol. 83:6115-6124, 2009). The recombinant virus ΔgM2, engineered not to express gM, produced a 3- to 4-fold decrease in viral titers and a 50% reduction in average plaque sizes in comparison to the HSV-1(F) parental virus. The recombinant virus containing all three mutations, gDΔct-ΔgM2-ΔgE, replicated approximately 1 log unit less efficiently than the HSV-1(F) parental virus and produced viral plaques which were on average one-third the size of those of HSV-1(F). The recombinant virus ΔUL11-ΔgM2, engineered not to express either UL11 or gM, replicated more than 1 log unit less efficiently and produced significantly smaller plaques than UL11-null or gM-null viruses alone, in agreement with the results of Leege et al. (T. Leege et al., J. Virol. 83:896-907, 2009). Analyses of particle-to-PFU ratios, relative plaque size, and kinetics of virus growth and ultrastructural visualization of glycoprotein-deficient mutant and wild-type virions indicate that gDΔct, gE, and gM function in a cooperative but not redundant manner in infectious virion morphogenesis. Overall, comparisons of single, double, and triple mutant viruses generated in the same HSV-1(F) genetic background indicated that lack of either UL20 or gK expression caused the most severe defects in cytoplasmic envelopment, egress, and infectious virus production, followed by the double deletion of UL11 and gM.
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