Document Type

Thesis - University Access Only

Award Date

2011

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

Abstract

The influenza A virus matrix protein M 1 is a multifunctional protein that plays numerous roles in viral replication including early entry, viral RNA replication, and assembly to the host cell. It forms a protein shell just beneath the lipid bilayer membrane and connects the viral transmembrane envelope proteins with the soluble vRNP complex. The Ml protein is a highly conserved 252-amino acid protein that contains two globular domains, an N terminal domain (residues 1-164) and a C-terminal domain (residues 165-252). Presence of an interdomain linker region that consists of 28 amino acid residues (residues 139-166) structurally connecting N- and C-domain of Ml protein has been suggested but its function is unknown. To study the potential role of this putative linker region in influenza A virus replication, we generated a panel of mutations targeting a four-residue segment (residues 163-166, RQMV) spanning the junction site between N- and C-domain of Ml protein and characterized their virus infectivity and other functional properties such as vRNA transcription and incorporation. We found that all mutations, regardless of whether they were conservative or non-conservative in nature, resulted in a replication-competent virus except for the M165A mutation. Further characterization of this mutant appeared to suggest that the M 165A mutation blocked influenza A virus replication by disrupting an entry entry event. Thus, our data provides evidence that the putative interdomain linker region of influenza A Matrix: 1 (M 1) protein is important for virus replication. M 1 C-terminal domain encodes another RQMV segment spanning residues 210-213, which is located in a putative helix 12 in the Ml protein. The same strategy was used to study the role of this 2nd RQMV segment in virus replication. Mutations in the second RQMV appeared to result in more diverse phenotypes compared to those targeting the first RQMV. Quantitative replication kinetic experiments discriminated mutant viruses into three groups: wild-type (WT)-like replication competent, replication attenuated, and completely replication defective. In WT-like replication competent group, Q211A and Q21 IN replicated as efficiently as WT, indicating mutations exerted no effect on virus replication. Most of the mutations resulted in viruses belonging to the replication attenuated group. These mutations are R210A, R210K, Q211E, M212A, and M212L, which showed an initial lag in replication kinetics but eventually reached titers comparable to those of WT viruses. In marked contrast, third group consisting of V213A and V213L was replication-incompetent. These results demonstrated for the first time a role of second RQMV segment and probably putative helix 12 in the C-terminal domain in influenza A virus replication. Future study is needed to explore mechanisms by which mutations cause defects in influenza A virus replication. A better understanding of these mechanisms is critical to elucidate and identify novel functions of influenza M 1 protein that can be used as new targets for anti-influenza drug therapy.

Library of Congress Subject Headings

Influenza A virus

Viruses -- Reproduction

Viral proteins

Format

application/pdf

Number of Pages

82

Publisher

South Dakota State University

Download

Share

COinS