A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration

Area of Science:

• Retroviral molecular biology
• Virology
• Molecular genetics

Background:

• Moloney murine leukemia virus (MuLV) is a retrovirus.
• Efficient integration of viral DNA into the host genome is crucial for retroviral replication.
• The pol gene encodes essential enzymatic activities for retroviral replication.

Purpose of the Study:

• To define a function encoded in the 3' region of the MuLV pol gene required for efficient viral DNA integration.
• To characterize a specific mutant (MuLV-SF1) affecting this function.

Main Methods:

• Site-directed mutagenesis of cloned MuLV DNA.
• Cotransfection of mutant DNA into rat cells with the herpes simplex virus thymidine kinase (HSV tk) gene.
• Production and infection with viral particles.
• DNA hybridization to assess viral DNA integration.
• Analysis of tk+ colony formation using pseudotypes.

Main Results:

• The MuLV-SF1 mutant, with an arginine to cysteine change in a conserved pol gene region, produced viral particles with reverse transcriptase activity.
• Infection with MuLV-SF1 particles resulted in a >10-fold decrease in integrated viral DNA and a >300-fold decrease in infection efficiency.
• Pseudotypes formed with MuLV-SF1 proteins were >100-fold less effective in producing tk+ colonies, with aberrant proviral integration observed.

Conclusions:

• The MuLV-SF1 pol gene is defective in a function essential for normal integrative recombination.
• This integration function is dissociable from viral DNA synthesis.
• The 3' region of the pol gene encodes a critical determinant for efficient and accurate retroviral integration.