ABSTRACT
Hepatitis C virus (HCV) is a positive strand RNA virus with certain similarity to flaviviruses and pestiviruses. To examine the processing and possible assembly of HCV proteins, we constructed a recombinant vaccinia virus that expresses a full-length genomic RNA, infected chimp liver cells with the virus, and analyzed HCV-related protein products by immunofluorescent antibody staining and Western blot detection with mouse monoclonal antibodies. The putative core, envelope, and NS1 and NS3 proteins that yielded from this recombinant were 22, 32, 53 to 58, and 65 kDa in size, respectively. The NS4 protein was unexpectedly small, with an estimated molecular weight of 7 kDa, and the NS5 protein was found to be further cleaved into 52-kDa NS5a and 58-kDa NS5b proteins, the latter of which contains a hallmark of RNA replicase. A point mutation in the putative protease domain of NS3 resulted in a failure in the production of NS3, NS4, NS5a, and NS5b, but coexpression of NS3 restored the proper processing of these proteins, demonstrating that NS3, the putative viral protease, is essential for the production of these nonstructural proteins. Thus, HCV strikingly resembles pestiviruses in the size and the processing mode of the nonstructural proteins, particularly NS4 and NS5.
Subject(s)
Endopeptidases/metabolism , Genes, Viral/genetics , Hepacivirus/enzymology , Hepacivirus/metabolism , Protein Processing, Post-Translational , Viral Nonstructural Proteins/biosynthesis , Viral Nonstructural Proteins/metabolism , Animals , Antibodies, Monoclonal , Antibodies, Viral , Base Sequence , Cells, Cultured , DNA Helicases/genetics , DNA Helicases/metabolism , Endopeptidases/genetics , Escherichia coli/genetics , Genome, Viral , Hepacivirus/genetics , Humans , Liver/cytology , Molecular Sequence Data , Pan troglodytes , Point Mutation , Protein Precursors/metabolism , Recombinant Proteins/biosynthesis , Transfection , Vaccinia virus/genetics , Viral Nonstructural Proteins/geneticsABSTRACT
In order to clone hepatitis C (blood-borne non-A, non-B hepatitis) virus, lambda gt11-cDNA library was constructed from RNA extracted from 100 liters serum collected from 1,047 donors with elevated ALT levels and negative for hepatitis B virus-DNA. The library was immunoscreened on Y1090 cells with pooled serum obtained from patients with acute hepatitis C or chronic hepatitis C. By screening 29 clones specific for Japanese hepatitis C infection were isolated. The specificity of these clones for hepatitis C infection was determined by panels constructed in 3 laboratories. Of these, 12 clones were specific for American hepatitis C infection as well. The nucleotide sequence (201 bp) of one of them was determined to be unique compared to known human viruses including hepatitis A virus, hepatitis B virus and hepatitis D virus. Southern blot analysis showed the absence of the sequence of the human genome in the clone. The predicted amino acid sequence is rich in residues of lysine, arginine, glutamic acid and asparagine, while lacking leucine, cysteine and methionine.
Subject(s)
Cloning, Molecular , DNA, Viral/genetics , DNA/genetics , Hepatitis C/genetics , Hepatitis, Viral, Human/genetics , RNA, Viral/blood , Acute Disease , Amino Acid Sequence , Base Sequence , Chronic Disease , Hepatitis C/blood , Humans , Molecular Sequence Data , RNA, Viral/analysis , Sequence Homology, Nucleic AcidABSTRACT
A lambda gt11-random-primed-cDNA clone specific for chronic hepatitis C was isolated from pooled serum presumably infected by hepatitis C virus. The translation product of the clone detect 50% of patients with chronic hepatitis C in 4 test panels but none of the patients with acute hepatitis C, other liver diseases or normal controls was positive for the peptide. The nucleotide sequence of the cDNA clone, the size of which is 66 bp, has no homology to the complete sequences of known human viruses such as adenovirus, coxsackievirus, rhinovirus, immunodeficiency virus type 1, Epstein-Barr virus, polioma virus, poliovirus, papilloma virus, parvovirus, papovavirus, varicella-zoster virus, yellow fever virus, endogenous retrovirus, T-cell lymphotropic virus types I, II, and III Japanese encephalitis virus, and hepatitis A, B, and D viruses. Probably only one or two epitopes are present on the molecule encoded by the clone as the peptide consists of only 22 amino acid residues.
