Genetically modified VSV(NJ) vector is capable of accommodating a large foreign gene insert and allows high level...

Area of Science:

• Virology
• Molecular Biology
• Gene Therapy

Background:

• Vesicular stomatitis virus (VSV), particularly Indiana serotype (VSV(Ind)), is utilized as a gene expression vector.
• The New Jersey serotype (VSV(NJ)) has seen less application as a gene expression vector.
• Developing RNA virus vectors for high-level expression of large foreign genes is crucial.

Purpose of the Study:

• To construct and characterize a genetically modified VSV(NJ) vector capable of accommodating and expressing large human hepatitis C virus (HCV) non-structural (NS) protein genes.
• To evaluate the expression levels and processing of HCV NS proteins within the modified VSV(NJ) vector system.
• To assess the capacity of the modified VSV(NJ) vector for foreign gene insertion and its impact on viral characteristics.

Main Methods:

• Genetic modification of the VSV(NJ) M gene (rVSV(NJ)-M) by altering specific methionine residues.
• Insertion of various lengths of HCV non-structural polyprotein genes into the rVSV(NJ)-M vector backbone using reverse genetics.
• Characterization of recombinant viruses via electron microscopy to determine viral length and diameter.
• Analysis of interferon-beta (IFN-β) induction in mouse fibroblast cells upon infection with wild-type and mutant VSV(NJ) viruses.
• Assessment of NS protein expression levels in both IFN-competent and IFN-incompetent cell lines.

Main Results:

• Recombinant VSV(NJ)-M vectors successfully accommodated up to 6000 bases of foreign HCV NS genes.
• Viral length increased proportionally with the size of inserted foreign genes (approx. 10 nm per 1kb).
• High-level expression and proper processing of all tested HCV NS proteins were confirmed.
• The rVSV(NJ)-M mutant virus induced higher IFN-β levels compared to wild-type VSV(NJ).
• NS protein expression was higher in IFN-incompetent cells than in IFN-competent cells.

Conclusions:

• The modified rVSV(NJ)-M vector is a robust platform for expressing large foreign genes, including complex viral polyproteins.
• The vector system facilitates efficient expression and processing of HCV NS proteins, with potential applications in virology research.
• The rVSV(NJ)-M mutant exhibits enhanced immunogenicity and expression capabilities, making it a promising tool for gene delivery and expression studies.