The goal of this application is to provide continuing support for Dr. Veronica Sanchez' training as a research scientist in the field of herpesvirology. Phase I of the proposed work will cultivate her research and teaching skills in a mentored environment in the laboratory of Dr. Deborah Spector at UCSD. Phase II will mark her transition to an independent research position in academia. Microtubule-based transport plays an important role in many cellular processes. Cytoplasmic dynein is a multimeric protein that, in association with the dynactin complex, mediates the transport of membranous and non-membranous cargo toward the microtubule-organizing center (MTOC) at the centrosome. Because the site of human cytomegalovirus (HCMV) envelopment overlaps the MTOC, we propose that dynein is involved in the transport of nucleocapsids from the nucleus to the cytoplasmic assembly compartment. To address this hypothesis, the expression and localization of dynein and dynactin subunits in HCMV-infected fibroblasts will be established. In addition, a functional analysis of dynein activity will be undertaken. Dynein activity will be inhibited in HCMV-infected cells by over-expression of dynactin subunits or small interfering RNAs (siRNAs) from recombinant viruses. The effects of dynein inhibition on the transport of nucleocapsids to the cytoplasmic assembly compartment will be determined by electron microscopy and immunofluorescence assays. The regulation of dynein activity by the cyclin-dependent kinases (cdks) in infected cells will also be studied. Recombinant HCMV viruses expressing dominant-negative forms of the cdks or siRNAs targeting the cyclins will be used to study the effect of phosphorylation on dynein activity in infected cells. These studies should provide valuable information regarding the role of dynein and cdk activity in HCMV assembly. In addition, the results from these studies will provide the basis for Dr. Sanchez to develop an independent research program and assist her to secure a faculty position at a university or medical school.
|Sanchez, Veronica; Mahr, Jeffrey A; Orazio, Nicole I et al. (2007) Nuclear export of the human cytomegalovirus tegument protein pp65 requires cyclin-dependent kinase activity and the Crm1 exporter. J Virol 81:11730-6|
|Sanchez, Veronica; Spector, Deborah H (2006) Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles. J Virol 80:5886-96|