Protein function is dynamically controlled by post-translation events. A connmon regulatory mechanism is the phosphorylation and subsequent conformational rearrangement of target proteins. Viral proteins are often controlled by the same pathways that target proteins encoded by the host. The replication cycle of influenza A virus is influenced by host proteins and provides a unique system in which to study these processes. A major target of regulation is the influenza virus replication machinery containing viral RNA, nucleoprotein (NP), and the trimeric polymerase composed ofthe proteins PB1, PB2, and PA. The replication machinery controls the ordered transition from gene expression to genome replication that is essential for a productive infection. In addition, the viral polymerase is a key determinant in the host range of influenza virus and restricts the transmission of influenza virus from avian to human populations. The processes controlling the replication machinery and the interplay between the polymerase and the host are poorly understood. We propose an integrative approach using biochemical, genetic, and structural studies to determine the host proteins and molecular mechanisms that regulate the influenza replication machinery. Specifically, Aim 1 will identify sites of phosphorylation within viral proteins and examine the functional consequences of phosphorylation on virus replication and the determination of host range.
Aim 2 will build on recent genetic mapping of interactions to produce high-resolution crystal structures that characterize in detail the protein interfaces of the replication complex. Finally, Aim 3 will exploit loss-of-function screens to identify cellular proteins that regulate the influenza polymerase and control transmission of influenza from birds to humans. These studies will provide crucial insight into the viral and host factors controlling influenza replication and will provide the foundation for rational strategies to treat and prevent future influenza outbreaks in humans.

Public Health Relevance

The influenza virus replication machinery is a key player in establishing infection and determining its pathogenicity. These studies will provide insight into the regulation ofthe influenza replication machinery, how it cpntrols transmission of influenza virus from birds to humans, and may ultimately identify new targets for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Transition Award (R00)
Project #
5R00GM088484-05
Application #
8601103
Study Section
Special Emphasis Panel (NSS)
Program Officer
Sakalian, Michael
Project Start
2009-09-07
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
5
Fiscal Year
2014
Total Cost
$263,912
Indirect Cost
$83,723
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Poole, Daniel S; Yú, Shu?qìng; Caì, Yíngyún et al. (2014) Influenza A virus polymerase is a site for adaptive changes during experimental evolution in bat cells. J Virol 88:12572-85
Tran, Vy; Moser, Lindsey A; Poole, Daniel S et al. (2013) Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread. J Virol 87:13321-9