Dan Wang

Document Type

Thesis - University Access Only

Award Date

2011

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

Abstract

New antiviral drugs are needed in combating future influenza pandemics. Therefore, a better understanding of influenza virus replication at the molecular level is important to identify new viral targets and develop novel antiviral therapies. One of the least understood parts of the influenza virus replication cycle is virus assembly and release, the step during which new virus particles emerge from the infected-cell surface and are released to spread the infection to new cells. The goal of this dissertation was two-fold. The first was to elucidate protein-protein interactions involving the formation of a heterotrimeric viral RNA polymerase complex that is composed of three subunits: polymerase basic protein 1 (PB 1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). The second was to study a role of the matrix 1 (M 1) protein in influenza virus particle formation. The influenza virus polymerase complex, consisting of the PA, PB 1, and PB2 subunits, is required for the transcription and replication ofthe influenza A viral genome. Previous studies have shown that PB 1 serves as a core subunit to incorporate PA and PB2 into the polymerase complex by directly interacting with PA and PB2. Despite numerous attempts, largely involving biochemical approaches, a specific interaction between PA and PB2 subunits has yet to be detected. In influenza virus polymerase project, we developed and utilized bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in the assembly of the influenza A virus polymerase complex. Proof-of-concept experiments demonstrated that BiFC can specifically detect PA-PB 1 interaction in living cells. Strikingly, BiFC demonstrated an interaction between PA and PB2 that has not been reported previously. Deletion-based BiFC experiments indicated that the N-terminal 100 amino acid residues of PA are responsible for the PA-PB2 interaction observed in BiFC. Furthermore, a detailed analysis of subcellular localization patterns and temporal nuclear import of PA-PB2 binary complexes suggested that PA and PB2 subunits interacted in the cytoplasm initially and were subsequently transported as a dimer into the nucleus. Taken together, results of our studies revealed a previously unknown PA-PB2 interaction and provided a frame work for further investigation of the biological relevance of PA-PB2 interaction in the polymerase activity and viral replication of influenza A virus. Part of the polymerase assembly research described in the dissertation has been published in J. Virol. 2009 83: 3944-3955(32). The matrix protein (M 1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M 1 into virus particles. In the influenza virus Ml project, we explored the mechanisms that control Ml budding. Similar to previous studies, we found that Ml by itself fails to form virus-like-particles (VLPs). We further demonstrated that Ml, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane where influenza virions bud. Remarkably, we showed that a I 0-residue membrane targeting peptide from either the Fyn or Lek oncoproteins appended to Ml at the N-terminus redirected Ml to the plasma membrane and allowed MI particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M 1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 not mutant M2 effectively targeted Ml to the plasma membrane and produced extracellular Ml VLPs. Our results suggest that influenza MI may not possess an inherent membrane-targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M 1 in budding. This study highlights the fact that interactions of MI with viral envelope proteins are essential to direct MI to the plasma membrane for influenza particle release. Part of the polymerase assembly research described in the dissertation has been published in Journal of Virology. 2010 84: 4673-4681(93). In summary, our data provides strong evidence that multiple protein-protein interactions occur in influenza virus assembly and release. These intraviral protein-protein interactions are essential to virus replication and any disruption of such interactions spatially and temporarily will abrogate influenza virus infectivity. Further investigation of protein-protein interactions reported in this dissertation may aid in the elucidation of novel therapeutic targets that could be explored in the design of new generation of anti-influenza drugs that inhibit virus assembly and subsequently block influenza virus replication.

Library of Congress Subject Headings

Influenza A virus

Viruses -- Reproduction

Viral proteins

Format

application/pdf

Number of Pages

99

Publisher

South Dakota State University

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