The long term objectives of this research program are to define the mechanisms of fundamental replication processes common to many important (+) strand RNA viruses, to determine the causes of virus-induced cytopathology, and to use these results to develop more effective antiviral strategies and enhance the beneficial use of RNA viruses and their components in genetic engineering and medicine. New directions in studies of virus-encoded functions in RNA replication, gene expression and encapsidation will be addressed using brome mosaic virus (BMV), a representative member of the alphavirus-like superfamily and a productive, advanced model system. Unique, new opportunities to study these viral functions have been created by recent studies of the functions and interactions of the heliease-like 1a and polymerase-like 2a BMV RNA replication proteins and their BMV RNA templates, and by recent demonstrations that BMV directs RNA-dependent RNA replication, mRNA transcription, and virion assembly in the experimentally tractable yeast, S. cerevisiae. This BMV/yeast system and complementary genetic, biochemical, and cell biology approaches will be used to define and analyze successive steps in assembly and function of the RNA replication/transcription complex. In vivo and in vitro tests will be used to analyze why in vivo assembly of the BMV RNA-dependent RNA polymerase complex has a snRNP- or ribosome- like requirement for viral RNA, to test indications that (-) strand initiation depends on replicase interaction with at least two widely separated sites on the template RNA, and to explore the initiation of (+) strand RNA synthesis. The role of 1a-2a interactions in specific steps of RNA synthesis will be studied. Immunolabeling coupled with confocal and electron microscopy will be used to define the structure and organization of the membrane-bound RNA replication complex and the induction of membrane vesicles associated with this complex. Yeast mutants will be used to determine how replication-associated vesicles are related to the secretory pathway. New approaches to analyze bromovirus encapsidation in vivo will be used to identify viral RNA encapsidation signals, and interactions between the highly based N-terminal capsid protein segment and viral RNAs will be studied.
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