Tomoko Date

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.

Although hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis, its pathogenic role in fulminant hepatitis remains controversial. A 32-year-old man contracted hepatitis. Serum ALT concentration was reached to 6,970 IU/L, the lowest prothrombin time value was 16% and jaundice and stage II encephalopathy were developed. HCV RNA was detected in this patient by reverse transcription polymerase chain reaction in sera at the acute phase, and it was undetectable during the remission phase when anti-HCV was found. The entire genome of infected HCV was recovered, cloned, and sequenced from this patient, and compared with the clones of six other chronic hepatitis patients. Phylogenetic analysis revealed a clustering around genotype 2a and a deviation from the other 2a chronic hepatitis strains. Calculating the genetic distance in each subgenomic region revealed that the 5'untranslated region (5'UTR), core, nonstructural (NS) 3, and NS5A were severely deviated. Of 20 clones of the hypervariable region (HVR), 17 showed an identical sequence with the others showing a difference of only one amino acid. HCV was isolated from a fulminant hepatitis patient and its entire genome was recovered; a clustering around genotype 2a was observed, but the sequence deviated especially in 5'UTR, core, NS3, and NS5A; and monoclonality of the HVR sequence was found not only in the fulminant hepatitis patient but in a certain percentage of chronic hepatitis patients.