Molecular cloning and characterization of hepatitis C virus / Meng-Che Yeh.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Yeh, Meng-Che.

Contributor:

Mason, William S., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Pathology.

Pathology--Penn dissertations.

Pathology.

Academic Dissertations as Topic.

Medical Subjects:

Pathology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Pathology.

Pathology--Penn dissertations.

Physical Description:

xi, 160 pages : illustrations ; 29 cm

Production:

1997.

Summary:

Hepatitis C Virus (HCV) is the major causative agent of Non-A, Non-B Hepatitis (NANBH) worldwide. It may cause both acute and chronic hepatitis and progress to cirrhosis and hepatocellular carcinoma. No effective vaccine against HCV has been developed and the current clinically used therapeutic agents are unsatisfactory. For a better understanding of viral genome replication, as a basis for development of new antiviral therapies we therefore attempted to establish a cell culture system that could support part or all of the viral life cycle.

We first obtained a full-length HCV cDNA and various deletion variants through molecular cloning. Sequencing analysis on both strands revealed that there were no deletions, insertions, and substitutions causing early termination of the open reading frames. The catalytic domains of nonstructural proteins and cleavage sites between individual proteins were shown to be conserved. In addition, sequence comparison with other strains showed that our isolate is classified as genotype I(1a).

The genomic RNA of HCV from infectious serum was characterized by Northern blot analysis and a 9.5 kb genomic RNA was identified by a plus-strand specific riboprobe. It was estimated by Northern analysis that the serum showing HCV-positivity after one round of PCR contains ca. 10$\sp4$ to 10$\sp5$ genomes per ml of serum.

Results of proteins expressed in vitro from the cloned deletion variants confirmed the previous reports that the first 10 codons in core protein are important for optimal internal ribosomal entry. We also demonstrated that the cloned HCV variants are capable of encoding full-length polyproteins. Furthermore, evidence implied that the NS3 fusion protein encoded by HCVD2 is a functional viral protease and is able to process itself from the N-terminus of NS4A protein and to further process other nonstructural proteins.

Finally, in cell culture experiments we detected minus-strand HCV RNA after transfection with plus-strand HCVFL RNA. Minus-strand RNA was detected at 1 week and 2 weeks in HCVFL RNA transfected cells, and in HCVD3 RNA transfected cells at 2 weeks, which indicates that our cell culture system, after transfection with HCVFL and HCVD3 RNAs, may have transiently supported HCV RNA synthesis. Neither plus- nor minus-strand RNA was detected in any cultures by 4 weeks. This system provides us with a useful tool for the future studies of viral genome replication and antiviral agent screening.

Notes:

Supervisor: William S. Mason.

Thesis (Ph.D. in Pathology) -- University of Pennsylvania, 1997.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 98-00943.

OCLC:

244970599