The exceptionally broad tissue tropism of human cytomegalovirus (CMV) is a substantial contributor to the severe multi-organ disease caused by this virus in immune compromised individuals. Virtually all cell types, with the exception of lymphocytes and polymorph nuclear leukocytes, can support viral replication in vivo. Despite the importance of this virus'wide tropism in the development of pathogenesis, the molecular mechanisms mediating viral entry into a variety of different cell types, and the identity and function of the proteins involved are still poorly defined. The goal of this proposal is to determine whether a new viral protein recently identified by our group is a mediator of CMV tropism for epithelial (EPC), endothelial (EC) and dendrite cells (DC), three cell types that play fundamental roles in virus entry, dissemination and immunoevasion during infection in vivo. To identify new viral proteins potentially involved in mediating virus infection of specific cell types, the coding content of the broadly tropic strain TB40/E was compared to that of Towne, a strain with highly limited tropism. A putative open-reading frame (ORF) predicted to encode a protein of 97 aa in TB40/E and of 176 aa in Towne was selected for further analyses, based on the hypothesis that the longer isoform encoded by Towne might be non-functional. Polyclonal antibodies raised against residues 21-39 of the predicted amino acids sequence revealed that this ORF was indeed expressed during infection, and that the encoded protein localized to the nuclear rim at late times post-infection. Based on this specific sub cellular localization, we named this gene product nuclear Rim Associated CytomegalovirAL protein, or RASCAL. No difference was observed in the intracellular localization of RASCALTB40/E and RASCALTowne, and both isoforms remained associated with lamin B-positive cytoplasmic vesicles during virion egress from the nucleus. Recently completed immunoblot analyses of purified virions further revealed that both proteins might be contained in mature viral particles. We thus speculate that, as a virion component, RASCAL might promote infection of specific cell types by enhancing trafficking of penetrated virions towards the nucleus, or the nuclear deposition of the viral genome during entry, two critical steps for the establishment of productive infections in EPC, EC and DC. We also suggest that implementation of these functions might be hampered by the presence of the additional 79 amino acids in the longer RASCAL isoform, thus limiting the tropism range of viral strains encoding it. In this proposal we seek to determine if RASCAL is a tegument protein, and if it plays a role in viral tropism for EPC, EC and DC.
Cytomegalovirus (CMV) infections are a serious source of morbidity and mortality for immune compromised individuals such as AIDS patients, transplant recipients, and newborns. Because of this, the development of novel strategies to block viral infection and immune-suppression is a priority. Our analyses will establish if a newly identified protein encoded by CMV is involved in mediating viral infection of epithelial, endothelial and dendrite cells, three highly relevant cell types for viral proliferation, dissemination and immunoevasion in vivo. If successful, this research will provide a new target for the design of improved inhibitors of viral dissemination and infection-associated tissue damage.
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