PMID- 38230952 OWN - NLM STAT- MEDLINE DCOM- 20240208 LR - 20240210 IS - 2165-0497 (Electronic) IS - 2165-0497 (Linking) VI - 12 IP - 2 DP - 2024 Feb 6 TI - Landscape of protein-protein interactions during hepatitis C virus assembly and release. PG - e0256222 LID - 10.1128/spectrum.02562-22 [doi] LID - e02562-22 AB - Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites. FAU - Matthaei, Alina AU - Matthaei A AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. FAU - Joecks, Sebastian AU - Joecks S AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. FAU - Frauenstein, Annika AU - Frauenstein A AD - RG Experimental Systems Immunology, Max-Planck Institute for Biochemistry, Planegg, Bavaria, Germany. FAU - Bruening, Janina AU - Bruening J AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. FAU - Bankwitz, Dorothea AU - Bankwitz D AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. FAU - Friesland, Martina AU - Friesland M AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. FAU - Gerold, Gisa AU - Gerold G AUID- ORCID: 0000-0002-1326-5038 AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. AD - Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany. AD - Department of Clinical Microbiology, Virology, Umea University, Umea, Sweden. AD - Wallenberg Centre for Molecular Medicine (WCMM), Umea University, Umea, Sweden. FAU - Vieyres, Gabrielle AU - Vieyres G AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. AD - Junior Research Group "Cell Biology of RNA Viruses," Leibniz Institute of Experimental Virology, Hamburg, Germany. FAU - Kaderali, Lars AU - Kaderali L AD - Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany. FAU - Meissner, Felix AU - Meissner F AUID- ORCID: 0000-0003-1000-7989 AD - RG Experimental Systems Immunology, Max-Planck Institute for Biochemistry, Planegg, Bavaria, Germany. AD - Systems Immunology and Proteomics, Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany. FAU - Pietschmann, Thomas AU - Pietschmann T AUID- ORCID: 0000-0001-6789-4422 AD - Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Lower Saxony, Germany. LA - eng GR - 158989968/Deutsche Forschungsgemeinschaft (DFG)/ GR - 158989968/Deutsche Forschungsgemeinschaft (DFG)/ GR - Knut och Alice Wallenbergs Stiftelse (Knut and Alice Wallenberg Foundation)/ GR - 390874280/Deutsche Forschungsgemeinschaft (DFG)/ PT - Journal Article PT - Review DEP - 20240117 PL - United States TA - Microbiol Spectr JT - Microbiology spectrum JID - 101634614 RN - 0 (Viral Nonstructural Proteins) RN - 0 (Proteome) RN - 0 (Apolipoproteins E) RN - 0 (Apolipoproteins) RN - 156533-33-4 (RAD23A protein, human) RN - 0 (DNA-Binding Proteins) RN - EC 6.5.1.- (DNA Repair Enzymes) SB - IM MH - Humans MH - *Hepacivirus/genetics MH - Viral Nonstructural Proteins/genetics MH - Proteome/metabolism MH - Cell Line MH - *Hepatitis C MH - Apolipoproteins E/metabolism MH - Apolipoproteins/metabolism MH - DNA-Binding Proteins/metabolism MH - DNA Repair Enzymes/metabolism PMC - PMC10846047 OTO - NOTNLM OT - ERAD OT - HSPA5 OT - Rad23B OT - affinity purification OT - endoplasmic reticulum OT - hepatitis C virus OT - host-pathogen interactions OT - lipoproteins OT - proteomics OT - viral assembly and release OT - virus-host interactions COIS- The authors declare no conflict of interest. EDAT- 2024/01/17 12:43 MHDA- 2024/02/08 06:42 PMCR- 2024/01/17 CRDT- 2024/01/17 09:03 PHST- 2024/02/08 06:42 [medline] PHST- 2024/01/17 12:43 [pubmed] PHST- 2024/01/17 09:03 [entrez] PHST- 2024/01/17 00:00 [pmc-release] AID - 02562-22 [pii] AID - spectrum.02562-22 [pii] AID - 10.1128/spectrum.02562-22 [doi] PST - ppublish SO - Microbiol Spectr. 2024 Feb 6;12(2):e0256222. doi: 10.1128/spectrum.02562-22. Epub 2024 Jan 17.