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Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro.

ABSTRACT: Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5' untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.

SUBMITTER: Sugiyama K

PROVIDER: S-EPMC2698526 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro.

Sugiyama Kazuo K Suzuki Kenji K Nakazawa Takahide T Funami Kenji K Hishiki Takayuki T Ogawa Kazuya K Saito Satoru S Shimotohno Kumiko W KW Suzuki Takeshi T Shimizu Yuko Y Tobita Reiri R Hijikata Makoto M Takaku Hiroshi H Shimotohno Kunitada K

Journal of virology 20090415 13

Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5' untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a ...[more]

PMID: 19369330

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Project description:Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly.IMPORTANCE HCV DVGs have been identified in vivo and in vitro, but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.

Dataset Information

Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro.

Publications

Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro.

Similar Datasets

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