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Intraviral interactome of Chikungunya virus reveals the homo-oligomerization and palmitoylation of structural protein TF.

ABSTRACT: Chikungunya virus (CHIKV) is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain. Although the protein-protein interactions (PPIs) between nonstructural proteins of CHIKV have been extensively established, the complete CHIKV intraviral interactome remains to be elucidated. In this study, we examined all possible CHIKV intraviral PPIs by immunoprecipitation and constructed the intraviral interactome of CHIKV. We reported 19 novel PPIs including the homo-oligomerization of TF. Disulfide bonds promoted the oligomerization of CHIKV TF protein. 2-BP, a palmitoylation inhibitor reduced the palmitoylation of TF and increased TF oligomerization. A quadruple mutant of Cys33, Cys35, Cys41, and Cys43 in TF blocked its palmitoylation and reduced oligomerization. Furthermore, we determined the association of TF with nsP1 and nsP3 in a palmitoylation-dependent manner. Construction of intraviral interactome of CHIKV provides the basis for further studying the function of CHIKV proteins.

SUBMITTER: Yin H

PROVIDER: S-EPMC7092876 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Intraviral interactome of Chikungunya virus reveals the homo-oligomerization and palmitoylation of structural protein TF.

Yin Han H Yin Peiqi P Zhao Hongjian H Zhang Na N Jian Xia X Song Siqi S Gao Shan S Zhang Leiliang L

Biochemical and biophysical research communications 20190417 4

Chikungunya virus (CHIKV) is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain. Although the protein-protein interactions (PPIs) between nonstructural proteins of CHIKV have been extensively established, the complete CHIKV intraviral interactome remains to be elucidated. In this study, we examined all possible CHIKV intraviral PPIs by immunoprecipitation and constructed the intraviral interactome of CHIKV. We reported 19 novel PPIs including the homo-oligomerization of T ...[more]

PMID: 31005258

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Project description:Protein interactions are major driving forces behind the functional phenotypes of biological processes. As such, evolutionary footprints are reflected in system-level collections of protein-protein interactions (PPIs), i.e., protein interactomes. We conducted a comparative analysis of intraviral protein interactomes for representative species of each of the three subfamilies of herpesviruses (herpes simplex virus 1, human cytomegalovirus, and Epstein-Barr virus), which are highly prevalent etiologic agents of important human diseases. The intraviral interactomes were reconstructed by combining experimentally supported and computationally predicted protein-protein interactions. Using cross-species network comparison, we then identified family-wise conserved interactions and protein complexes, which we defined as a herpesviral "central" intraviral protein interactome. A large number of widely accepted conserved herpesviral protein complexes are present in this central intraviral interactome, encouragingly supporting the biological coherence of our results. Importantly, these protein complexes represent most, if not all, of the essential steps required during a productive life cycle. Hence the central intraviral protein interactome could plausibly represent a minimal infectious interactome of the herpesvirus family across a variety of hosts. Our data, which have been integrated into our herpesvirus interactomics database, HVint2.0, could assist in creating comprehensive system-level computational models of this viral lineage.IMPORTANCE Herpesviruses are an important socioeconomic burden for both humans and livestock. Throughout their long evolutionary history, individual herpesvirus species have developed remarkable host specificity, while collectively the Herpesviridae family has evolved to infect a large variety of eukaryotic hosts. The development of approaches to fight herpesvirus infections has been hampered by the complexity of herpesviruses' genomes, proteomes, and structural features. The data and insights generated by our study add to the understanding of the functional organization of herpesvirus-encoded proteins, specifically of family-wise conserved features defining essential components required for a productive infectious cycle across different hosts, which can contribute toward the conceptualization of antiherpetic infection strategies with an effect on a broader range of target species. All of the generated data have been made freely available through our HVint2.0 database, a dedicated resource of curated herpesvirus interactomics purposely created to promote and assist future studies in the field.

Project description:Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

Dataset Information

Intraviral interactome of Chikungunya virus reveals the homo-oligomerization and palmitoylation of structural protein TF.

Publications

Intraviral interactome of Chikungunya virus reveals the homo-oligomerization and palmitoylation of structural protein TF.

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