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Hiv Error Prone Polymerase

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Frequency of Mutations Made in a Single Cycle of HIV-1 Replication▿ † Michael E. Abram1, Andrea L. Ferris1, Wei Shao2, W. Gregory Alvord3 and Stephen H. Hughes1,* 1HIV http://jvi.asm.org/content/84/19/9864.full Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 2Advanced Biomedical Computing Center, SAIC-Frederick Inc., Frederick, Maryland 3Data Management Services, National Cancer Institute at Frederick, Frederick, Maryland  Next Section ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral error rate replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. hiv error prone We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro. Human immunodeficiency virus type 1 (HIV-1) is genetically diverse both within and between infected individuals (6, 12, 66). The high rate of viral replication, the size of the viral population in individual patients

 

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