Protein-protein interactions between hepatitis C virus nonstructural proteins.
Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.
Dimitrova
Maria
M
INSERM UMR_U544, Institut de Virologie, 67000 Strasbourg, France.
Imbert
Isabelle
I
Kieny
Marie Paule
MP
Schuster
Catherine
C
D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D018613:Microscopy, Confocal; D011485:Protein Binding; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
D017361:Viral Nonstructural Proteins