Oxidative base damage promotes mutations by substantially increasing the frequency of mispair formation during DNA replication. While much is known about how oxidative base damage contributes to mutations during the replication of genomic DNA, much less is known about the impact of oxidative base damage for replication of RNA. Oxidative base damage is likely to be of particular importance in the hypermutation of retroviruses such as HIV. This is because infection associated oxidative stress substantially increases nucleic acid base damage; RNA is not subject to repair; and replication is mediated by error prone reverse transcriptase. In the initial period of funding for this grant, we established that several oxidative lesions promote base substitutions at high frequencies during replication by HIV reverse transcriptase (HIV-RT). A primary focus of this continuation application is to further define the mechanisms whereby oxidative base damage of nucleic acids contributes to HIV hypermutation. We will develop an integrated mechanistic approach aimed at investigating factors which influence hypermutation in HIV. Kinetic methods will be used to examine the differential utilization of oxidized bases during the RNA and DNA template directed phases of retroviral replication. The influence of RNA template secondary structure and sequence context on oxidized base induced hypermutation will be determined. How retroviral accessory proteins which participate in replication modify these effects will be studied. The influence on retroviral mutation spectra resulting from RNA base damage caused by iron induced oxidative stress, peroxyl radicals, nitric oxide and peroxynitrite will be directly ascertained using a novel retroviral forward mutation assay.Oxidative base damage, a high frequency of retroviral replication mediated by error prone reverse transcriptase, template structure and composition, and additional protein factors contribute synergistically to the phenomenon of hypermutation in HIV. This results in viral escape from immune system surveillance and the rapid evolution of drug resistance in people with AIDS. Identifying factors that can be manipulated to attenuate hypermutation should allow for the development of strategies aimed at limiting the acquisition of drug resistance and improve the effectiveness of conventional antiviral therapies. Alternatively, strategies to increase the rate of hypermutation could increase the production of defective or non-viable virus, thus limiting the extent of active infection.
