Replicating foamy virus-based vectors directing high level expression of foreign genes

Area of Science:

• * Virology
• * Molecular Biology
• * Gene Therapy

Background:

• * Retroviral vectors are crucial tools for gene delivery and expression.
• * Human foamy virus (HFV) offers a unique platform for vector development due to its biological characteristics.
• * Modifications in the U3 region of the long terminal repeat and 3' genome region of HFV are nonessential for replication.

Purpose of the Study:

• * To construct and characterize novel replication-competent retroviral vectors based on a modified human foamy virus molecular clone.
• * To evaluate the efficiency of foreign gene expression using different vector designs.
• * To assess the utility of these vectors for studying protein functions in cell culture.

Main Methods:

• * Construction of pFOV vectors (pFOV-1 to -3 and -7) with deletions in nonessential viral regions.
• * Fusion of indicator genes (CAT, luciferase) to the Bet protein or use of foot-and-mouth disease 2A protease for self-cleavage.
• * Insertion of mouse hepatitis virus surface gene fragments into pFOV-1.
• * Infection of diploid human fibroblasts with constructed viruses and assessment of foreign protein expression.

Main Results:

• * Developed replication-competent retroviral vectors (pFOV) derived from infectious HFV molecular clone.
• * Vectors pFOV-1, -2, -3, and -7 demonstrated efficient expression of indicator genes and foreign proteins.
• * Recombinant viruses were infectious in human fibroblasts and stably expressed high levels of inserted foreign proteins.
• * Foot-and-mouth disease 2A protease enabled self-cleavage of fusion proteins in pFOV-7.
• * Internal ribosomal binding site allowed authentic foreign protein expression in pFOV-2 and -3.

Conclusions:

• * The pFOV vector system provides a robust platform for delivering and expressing foreign genes in mammalian cells.
• * These vectors are effective tools for studying the biological effects of proteins of interest in tissue culture.
• * The developed vectors hold potential for various applications in molecular biology and virology research.