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Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.

ABSTRACT: Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major products, named gp35 and gp70, were shown to be transported into microsomes and heavily glycosylated, suggesting that the processing events are partly mediated by the signal peptidase of the endoplasmic reticulum. The other two products, p19 and p21, were probably associated with the outer surface of the microsomal membrane. Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino-terminal amino acid sequences of gp35 and gp70 indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH2-p22-gp35-gp70-p19. Both gp35 and gp70 could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.

SUBMITTER: Hijikata M

PROVIDER: S-EPMC51914 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.

Hijikata M M Kato N N Ootsuyama Y Y Nakagawa M M Shimotohno K K

Proceedings of the National Academy of Sciences of the United States of America 19910701 13

Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major p ...[more]

PMID: 1648221

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Project description:BACKGROUND:Hepatitis C virus (HCV) is the cause of high morbidity and mortality worldwide, inflicting around one million people in Pakistan alone. The HCV genomic RNA harbors conserved structural elements that are indispensable for its replication. The 3' untranslated region (UTR) contains several of these elements essentially involved in regulating the major steps of the viral life cycle. OBJECTIVES:Differences in regulatory elements of HCV may contribute towards differential infectivity of local isolates. The present study explicates sequence analysis and secondary structure prediction of HCV 3'UTR region of subtype 3a from Pakistan to characterize this particular region. PATIENTS AND METHODS:HCV 3'UTR region was amplified, cloned and sequenced from five different patients. Sequence and structural analysis was performed and phylogenetic analysis was carried out using the 3'UTR sequence reported in NCBI nucleotide data base (http://www.ncbi.nlm.nih.gov/nuccore) by other studies. RESULTS:Sequence analysis of the amplified fragment from five patients indicated that the 3'UTR is composed of 214-235 nts. Its sequence contains a type-specific variable region followed by a poly U/UC region and a highly conserved X-tail of 98 nts. The variable region reported here has 26 nts and one stem loop at the secondary structure that differentiate it from HCV genotype 1a ( GT1a) 3'UTR which contains additional 14 nts and two stem loops. The poly U/UC region varied in length (100-79 nts) and nucleotide sequence within the Pakistani isolates, and among different genotypes. Some substitutions found in the X-tail do not affect secondary structure of this element suggesting that this region might play an important role in replication, stabilization and packaging of HCV genome. Additionally, U residues are not present at the end of the X-tail in Pakistani 3a isolates as otherwise reported for the variants of genotype 1b. CONCLUSIONS:Sequence and structural diversity of the 3'UTR variable region and Poly U/UC region found in the local isolates indicate specificity in the regulating elements of 3'UTR that might be associated with differential replication efficacy of the HCV Pakistani isolates. The study necessitates functional characterization of these regulating elements to elucidate variable viral efficiency and pathogenicity associated with inter-geographical isolates.

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Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.

Publications

Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.

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