Rahm Gummuluru, PhD
Associate Professor of Microbiology
BS, University of Saskatchewan
MS/PhD, University of Rochester School of Medicine
Postdoctoral Training, Fred Hutchinson Cancer Institute
We are using genetic, immunological and biochemical approaches to identify the molecular mechanisms of human immunodeficiency virus (HIV) – dendritic cell (DC) interactions, and the putative contributions of viral accessory genes to HIV-1 replication in DC- T cell co-cultures.
The research in my laboratory is broadly focused on the role of dendritic cells (DC) in the initiation and propagation of HIV-1 replication, and the mechanism of subversion of DC program by the virus. Since dendritic cells are believed to be the first immune competent cells to encounter virus in the genital mucosa, a thorough understanding of HIV-DC interactions is of
paramount importance. DC can capture virus particles independently of CD4 and co-receptor complexes, and retain them in an infectious state for an extended period of time. These virus-bearing DC may then facilitate a more efficient spread of virus to replication-permissive CD4+ T cells. DC-SIGN, a mannose binding C-type lectin receptor, is one virus-attachment factor that captures infectious virus particles, and facilitates trans-infection of CD4+ T cells. Our previous work has identified DC-SIGN independent mechanisms of virus attachment by DC. Hence, we are utilizing novel genetic screens to identify virus-capture mechanisms displayed by dendritic cells. The fate of the virus particle post-attachment, be it via DC-SIGN, or other molecules, in DC also remains unclear. Virion trafficking within DC also seems to bypass conventional endocytic organelles, i.e., endosomes and lysosomes. Virus localization within this novel vesicular compartment not only has the potential to protect the invading HIV from being degraded, but also creates a latent reservoir of virus that could present a major challenge for eradication by antiretroviral therapy. Furthermore, the mechanism of subsequent return of infectious virus particles to the cell surface and the method of virus transmission to T cells remains unclear. Current studies utilizing biochemical and microscopic approaches to delineate molecular pathways are underway to monitor HIV-1 trafficking and localization in the DC and its subsequent transfer to T cells. These studies will aid in our understanding of the mechanism of HIV transmission to the naïve host and might lead to the identification of novel therapies that prevent establishment of virus infection.
Amongst retroviruses, HIV replication is quite unique, in that the viral genome encodes a number of accessory and regulatory genes that distinguish them from the prototypical “simple” retroviruses. These accessory genes, vpu, vif, vpr and nef, though not required for virus replication in vitro, are essential for optimal viral fitness in vivo. Although a number of functions have been ascribed to the accessory genes of HIV, their exact role in virus replication has been difficult to ascertain due to a lack of a suitable in vitro replication system. In order to address this question, we have developed a DC – T cell co-culture system called the rapid turnover assay where the infected cells are subject to restrictions similar to that in vivo, namely the short half-life of infected CD4+ T cells. Virus strains that either express or are deficient in their ability to express one or more of the accessory genes of HIV-1 are competed against one another in a pair-wise fashion in this co-culture system. Contributions of accessory genes to virus fitness are then determined via a competitive replication analysis. These studies will allow us to determine the relevant functions of viral accessory genes. We are also using this assay to determine the relative fitness of mutant viral strains that arise in patients on therapeutic reverse transcriptase and protease inhibitors. Performing replication assays with virus strains in a cellular environment that is more representative of the in vivo situation will provide new insights into the pathogenesis of HIV-1 in vivo.