Rubella virus (RV) is a human pathogen and the causative agent of German measles. RV infection during the first trimester of pregnancy can cause fetal death or multisystem birth defects. Histopathological analysis of several tissues of infants with congenital rubella syndrome revealed generalized growth retardation, possibly due to mitotic inhibition. Growth retardation and other congenital effects due to RV infection have been reproduced in some animal model systems. Further, a mitotic inhibitory factor has been identified also from the RV-infected cells. To understand the mechanisms of RV induced growth inhibition, we wish to identify cellular factors that are responsible for cellular growth and analyze their role in RV replication. In addition, we want to understand the how natural RV infection or immunization alters the function of these growth factors. We have characterized the host-encoded proteins that interact with the cis-acting elements of RV RNA that are essential for replication. One of the host proteins that has been identified is a homologue of human calreticulin. Calreticulin is a phosphoprotein and is hyperphosphorylated upon RV infection. The phosphorylation site(s) has been localized in the N-terminal domain of calreticulin and its interaction with RV RNA is dependent upon the phosphorylation. Phosphorylation of calreticulin in uninfected cells is dependent on the state of cell cycle. Further, RV infection alters the state of cell cycle resulting in the acceleration of calreticulin phosphorylation. To define the role of calreticulin in RV replication, we have developed cell lines which over-express calreticulin. Preliminary results suggest that these cells support enhanced RV replication. To understand the role of phosphorylated calreticulin and the RV induced alteration of cell cycle, we are studying the interactions of calreticulin with RV replication proteins and cell cycle components. We have cloned the viral proteins as well as calreticulin, and are analyzing the interaction between these proteins in a """"""""two-hybrid system"""""""". In addition, we are also studying the association (in vivo and in vitro) between these proteins by immunoprecipitation and Western blot analysis. Characterization and identification of cellular factors that are necessary for viral pathogenesis may provide clues for the cause of RV vaccine associated complications and provide necessary information for the improvement of future RV and other childhood vaccines. Publication: N. K. Singh, C. D. Atreya, H. L. Nakhasi (1994) Proc. Natl. Acad. Sci. USA 91, 12770-12774. C. D. Atreya, N. K. Singh, H. L. Nakhasi (1995) J. Virol. 69, 3848-3851. C. D. Atreya, G. P. Pogue, N. K. Singh, H. L. Nakhasi (1995) In Structure, Function and Cell Biology of Calreticulin (Ed. M. Michalak) R. G. Landes, Co; Austin, TX (in Press).
