Anthony R. Dawson

Position title: Mehle Laboratory


3325 Microbial Sciences Building
1550 Linden Dr
Madison, WI 53706

Research Title.  Phospho-Regulation of the Influenza Virus Replication Machinery.

Research Description. Influenza viruses are the etiological agents for seasonal influenza outbreaks and sporadic influenza pandemics in both human and avian populations, resulting in high public health and economic burdens worldwide. The success of these viruses requires host factors to regulate both cellular and viral protein function. Anthony’s research focuses on how host proteins regulate the formation and function of the viral ribonucleoprotein (RNP) complex. The influenza RNP serves as the minimal unit required for viral gene expression and replication. Consequently, proper RNP formation is indispensable for influenza virus replication. The RNP is composed of the heterotrimeric viral polymerase bound to both ends of the genomic RNA that is coated by nucleoprotein (NP). During RNP generation, monomeric NP oligomerizes to encapsidate nascent RNA produced by the viral polymerase. Previous work from the Mehle lab has demonstrated that host PKC phosphorylates NP and regulates its oligomerization specifically controlling replication and de novo RNP assembly. Dynamic phosphorylation of NP is necessary for successful replication. Yet, how NP phosphorylation is temporally regulated to enable gene expression early during infection and RNP assembly and genome replication later in infection remains poorly understood. Anthony is carrying out a spatiotemporal analysis of NP phosphorylation. NP was purified from infected cells at multiple time points post-inoculation, and he is currently quantifying its phosphorylation status by mass spectrometry. Functional assays are in place to test the impact of any changing patterns in phosphorylation that are identified. Moreover, as phosphorylation blocks NP oligomerization, an intriguing possibility is that host phosphatase licenses NP incorporation into RNPs. Anthony is testing whether specific nuclear phosphatases control NP incorporation into RNPs. Recent structures of the influenza polymerase revealed that transcribing and replicating polymerases adopt distinct conformations. How the polymerase assumes conformations to promote transcription or replication remain unknown. Phosphorylation of the polymerase is known to occur, however discrete consequences are unknown. Preliminary data show that PKCδ phosphorylates the polymerase. Therefore, Anthony asked whether PKCδ phospho-regulates the polymerase, independent of its effect on NP function. Preliminary experiments measured the amount of transcription and replication products produced by the viral polymerase from a short viral template that does not require NP. Overexpression of PKCδ in these assays shifts polymerase function to produce a greater amount of replication products. This raises the possibility that PKCδ toggles the functions of the viral polymerase. This is an area Antony is actively pursuing. Secondly, mass spectrometry of the polymerase derived from the same conditions revealed a number of polymerase phospho-sites that are enriched or only present when PKCδ is overexpressed. The phosphorylated residues are located at functionally relevant regions of thepolymerase. Mimicking constitutive phosphorylation at a number of these sites inhibits polymerase activity. Anthony will assess each mutant polymerase’s ability to perform functions required for viral transcription and genome replication and to identify which residues are phosphorylated during infection. These experiments will provide a broad overview of the functional impacts of PKCδ on the function of the influenza virus RNP.

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