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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM035072-15
Application #
6018640
Study Section
Virology Study Section (VR)
Project Start
1985-07-01
Project End
2000-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
15
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Garcia-Ruiz, Hernan; Diaz, Arturo; Ahlquist, Paul (2018) Intermolecular RNA Recombination Occurs at Different Frequencies in Alternate Forms of Brome Mosaic Virus RNA Replication Compartments. Viruses 10:
Diaz, Arturo; Zhang, Jiantao; Ollwerther, Abigail et al. (2015) Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function. PLoS Pathog 11:e1004742
Hao, Linhui; Lindenbach, Brett; Wang, Xiaofeng et al. (2014) Genome-wide analysis of host factors in nodavirus RNA replication. PLoS One 9:e95799
Hao, Linhui; He, Qiuling; Wang, Zhishi et al. (2013) Limited agreement of independent RNAi screens for virus-required host genes owes more to false-negative than false-positive factors. PLoS Comput Biol 9:e1003235
Zhang, Jiantao; Diaz, Arturo; Mao, Lan et al. (2012) Host acyl coenzyme A binding protein regulates replication complex assembly and activity of a positive-strand RNA virus. J Virol 86:5110-21
Diaz, Arturo; Ahlquist, Paul (2012) Role of host reticulon proteins in rearranging membranes for positive-strand RNA virus replication. Curr Opin Microbiol 15:519-24
Gancarz, Brandi L; Hao, Linhui; He, Qiuling et al. (2011) Systematic identification of novel, essential host genes affecting bromovirus RNA replication. PLoS One 6:e23988
Wang, Xiaofeng; Diaz, Arturo; Hao, Linhui et al. (2011) Intersection of the multivesicular body pathway and lipid homeostasis in RNA replication by a positive-strand RNA virus. J Virol 85:5494-503
Kopek, Benjamin G; Settles, Erik W; Friesen, Paul D et al. (2010) Nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, RNA templates, and polymerase activity. J Virol 84:12492-503
Diaz, Arturo; Wang, Xiaofeng; Ahlquist, Paul (2010) Membrane-shaping host reticulon proteins play crucial roles in viral RNA replication compartment formation and function. Proc Natl Acad Sci U S A 107:16291-6

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