Analysis of RTs from other retroviruses in vitro suggests that HIV RT has exceptionally low fidelity. For example, MuLV-RT shows ~5-fold greater fidelity than HIV RT on both RNA and DNA templates across several publications from different groups, and even in identical experiments in the same publication. Yet cellular replication data indicates that these viruses have similar mutation rates. The mutation profile observed in cellular assay with HIV is also inconsistent with in vitro results. Recently, our lab and others have found that the in vitro fidelity of HIV RT improves when physiological concentrations of free Mg2+ (~0.25-1 mM) are used as opposed to the higher (5-10 mM) concentration optimized for in vitro activity that have typically been used in fidelity assays in vitro . This may be one factor, possibly among many, that explains differences between HIV RT and other viral RTs. MuLV and AMV RTs, for example, do not show significantly improved fidelity in lower Mg2+ and MuLV RT has an error rate in vitro that is more consistent with cellular replication values. The lack of correlation with respect to the overall mutation rate and spectrum of mutations observed in vitro vs. cellular replication has made it impossible to determine RT’s contribution to the mutation spectrum observed in cells. This in turn makes it difficult to assess the contributions of mutagenic cellular factors (e.g., APOBEC3G, UNG, and RNA polymerase II) to the observed spectrum. A thorough understanding of the factors that contribute to the genetic diversity of HIV will require a better understanding of HIV RT’s role in the process, and a clear definition of the mutation rate and spectrum observed with HIV RT in vitro, and during virus replication in cells. Understanding how HIV generates genetic diversity, which fuels the production of new viruses that can escape immunity and drug therapy, has been a long-standing goal of HIV researchers. We believe this proposal will move us forward toward this goal. New high fidelity Next Generation Sequencing (NGS) approaches, including Single Strand Consensus Sequencing (SSCS) and duplex sequencing, allow the rapid analysis of thousands of mutations in a single experiment and has brought in a new era for mutational analysis. The same can be said for recent findings from Dr. Wes Sundquist’s lab, our collaborator on this work, that demonstrate the complete replication of HIV dsDNA in a highly efficient cell-free virion-based endogenous reverse transcription (referred to as “ERT” in this proposal) system. By applying modern NGS approaches to DNA synthesis with purified RT in vitro, in ERT reactions, and during virus replication in cells, the mutation rates and spectrums of these processes can be directly compared. Notably, this will be the first fidelity analysis in the novel ERT system. This will more clearly define RT’s role and relative contribution to HIV mutagenesis, and the roles of virion and cellular factors. Specific Aims: Aim 1: To use Next Generation Sequencing (NGS) to compare the mutation rates and spectrums from purified HIV RT and highly efficient Endogenous Reverse Transcription (ERT) assays; Aim 2: To compare the rate and spectrum of mutations made by purified RT and in ERT reactions, to those observed during reverse transcription in cells.

Mutations lead to genetic diversity that fuels virus evolution which leads to viruses that escape drug therapy and the immune response. This work is relevant to human health because it will help define the sources of those mutations that arise during HIV replication, thus providing targets for future approaches to combating HIV.