Abstract
The role of Moloney murine leukemia virus (MoMLV) reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed. To that effect, the in vitro fidelity of the MoMLV RT was compared to the rate of base substitution mutations occurring during the replication of an MoMLV-based retroviral vector. Using the vector in an amber reversion assay, the base substitution mutation rate at a single locus was found to be 2 x 10(-6)/base pair in one cycle of vector virus replication. Analysis of the fidelity of the purified RT using the same template sequence revealed that, of the two mispairs (A.C and T.G) that would lead to reversion of the amber codon during replication, A.C occurs at a rate of 4.0 x 10(-6), and T.G occurs at a rate of 0.7 x 10(-4). While the rate of formation of A.C is very similar to the vector mutation rate, the rate of formation of T.G is more than 30 times higher. This discrepancy in rates suggests that there are other elements in the infected cells that contribute to the fidelity of viral replication.
Highlights
The role of Moloney murine leukemia virus (MoMLV)reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed
We found that tfhiedelity of plus strand synthesisby MoMLV R T in vitrowas about 30 times lower than theoverall mutation rateof the virus
Assay for MoMLV Mutation Rates during a Single Cycle of Replication-To determine the base substitutionmutation rate of MoMLV, an MoMLV-based vector system was developed that scored reversion mutations during a single cycle of viral replication (Fig. 1).This system is an extension of an assay previously used to measure spleen necrosis virus (SNV) mutation rate [7]
Summary
The role of Moloney murine leukemia virus (MoMLV)reverse transcriptase (RT) in the generation of base substitution mutations during retroviral replication was analyzed. Infected helper cell clones harboring a single provirus were isolated and used as source of vector virus for the determination of mutation rates.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
