title: A new cell-based assay for measuring the forward mutation rate of HIV-1.
abstract: Over 20 years into the ever-worsening AIDS pandemic, genetic variation remains the greatest obstacle for treating and preventing HIV-1 infection. Mutation rate assays for HIV-1 have been reported; however, none measure directly the forward mutation rate during replication of the virus in cell culture while still retaining the ability to propagate and further study mutant proviruses. Therefore, the objective of the current study was to develop such a phenotypic cell-based assay for measuring the forward mutation rate of HIV-1. Conventional recombinant DNA techniques and polymerase chain reaction were used to create a replication defective HIV-1 vector, pNL4-3Delta+cass, which is based on the NL4-3 strain and contains the thymidine kinase gene from human herpes virus type 1 as the mutational target. A series of transfection and infection steps were used to introduce the vector into 143B cells, which are negative for thymidine kinase function, and produce vector virus for a single cycle of replication. Viral titers were measured by counting the number of drug resistant colonies on the assay plates, and forward mutation rates were calculated from the viral titers. Mutant proviruses were sequenced to determine the types of genetic alterations that occurred. The average forward mutation rate for HIV-1 was 2.2 x 10(-5)mutations/base/cycle. The majority of mutations were base substitutions, including high frequencies of C-->U and G-->A transitions. Single adenosine insertions were also observed frequently. The new assay is economical and provides a direct measurement of the mutation rate during a single cycle of viral replication. Target cells containing mutant proviruses survive the drug selection process and may be propagated for further analysis. The new assay is novel and has many advantages over previous mutation rate assays and thus will be very useful in future studies on genetic variation of HIV-1.
authors.affiliation: Biomedical Sciences Ph.D. Program, Wright State University, Dayton, OH 45435, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Huang
authors.forename: D P
authors.initials: DP
authors.lastname: Wooley
mesh_terms: D002460:Cell Line; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D009154:Mutation