Professor Louis Mansky

These researchers are using MSI resources for two projects.

• HIV Reverse Transcriptase-Mediated Mutagenesis: HIV-1 has a high mutation rate, which contributes to its ability to evade the host immune system, limits the efficacy of antiretroviral drugs, and drives the emergence of drug resistance. Drug resistance conferring mutations as well as other viral mutations are primarily attributed to the error-prone nature of reverse transcriptase (RT). An intentional increase in RT-mediated mutations decreases virus infectivity by increasing the mutation rate to a level that is not able to maintain survival of the virus population. The potency by which HIV-1 infectivity can be decreased by increasing RT-mediated errors has led to an initiative to discover small molecules that may increase the HIV-1 mutation rate. An interdisciplinary collaborative team has been assembled to conduct discovery studies to identify new small molecules that increase RT-mediated errors, use molecular analyses to identify the mechanism(s) by which small molecules increase the HIV mutation rate and result in virus extinction, and to assess the mechanism of RT-mediated mutation using biochemical methods. Through preliminary studies, the researchers have identified four small molecules that increase RT-mediated mutations. In order to elucidate the structure-activity relationship driving this increase and to optimize this activity, they are first pursuing discovery studies to identify small molecules that can increase RT-mediated errors. The antiviral and mutagenic activities of these molecules will be assessed in cell culture. Second, they will examine the mechanism by which small molecules induce mutations and cause virus extinction in HIV-1 using cell culture methodologies. Here they will examine small molecules that they have already discovered as well as any lead molecules that they identify. Third, they will investigate the mechanism of action using biochemical methods to elucidate the mechanistic basis for increased RT-mediated mutation. Successful completion of these studies will provide deeper insight into the mechanisms of RT-mediated viral mutagenesis and its impact on viral replication and extinction.
• HTLV-1 Particle Analysis and Gag Interactions: Human T-cell leukemia virus (HTLV-1) infects about 20 million individuals worldwide and is the etiological agent of an adult T-cell leukemia/lymphoma (ATLL). It can also result in an inflammatory disease syndrome called HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). Prevalence rates for HTLV-1 infection in the general population are greater than 1% in the Caribbean Basin, Central Africa, and South Japan. HTLV-1 is notorious for being difficult to study in cell culture, which has prohibited a rigorous analysis of how these viruses replicate in cells, including the steps involved in retrovirus assembly. The details for how retrovirus particle assembly occurs are poorly understood even for other more tractable retroviral systems. Using a tractable model system, state-of-the-art biophysical approaches, and an interdisciplinary research team, these researchers have made novel observations that form the basis for this project. The researchers are beginning to investigate questions related to HTLV-1 particle size, Gag stoichiometry in particles, and HTLV-1 Gag interactions in living cells using multiple experimental approaches. In particular, they will apply cryo-electron microscopy/tomography (cryo-EM/ET), total internal reflection fluorescence (TIRF) microscopy, and the novel single-molecule technology of fluorescence fluctuation spectroscopy (FFS) to investigate questions related to particle size and Gag stoichiometry, Gag targeting to membrane, and HTLV-1 particle biogenesis. The results from these studies should provide further insight into fundamental aspects of HTLV-1 and retrovirus particle assembly, which may aid in developing therapeutics.