Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Microbiology and Microbial Pathogenesis

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Andrew Pekosz

Abstract

Influenza A virus encodes M2, a proton channel that has been shown to be important during virus entry and assembly. The primary aim of this thesis was to investigate the role of the membrane proximal region, residues 46-69, of the M2 cytoplasmic tail during virus replication. A cholesterol recognition/interaction amino consensus: CRAC) motif, previously identified in the membrane proximal region of M2 in some influenza A virus strains, was suggested to play a role in virus replication by mediating incorporation of M2 into budding virus particles. Alteration or completion of the M2 CRAC motif in two different recombinant virus strains caused no changes in virus replication in tissue culture; however, viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection. In order to further investigate the role of the membrane proximal residues of M2 in basic virus replication, scanning and directed alanine mutants were generated and analyzed in trans-complementation assays and recombinant viruses. The membrane proximal residues 46-69 tolerated numerous mutations with little, if any, affect on virus replication suggesting that the identity of individual amino acids in this region are less important than the overall protein structure for the M2 protein function. The requirement during virus replication of the ectodomain and the cytoplasmic tail of M2, which includes the membrane proximal region, was further characterized using the influenza C virus CM2 protein and a chimeric influenza A virus M2 protein: MCM) containing the CM2 transmembrane domain. While M2, CM2, and MCM could all alter cytosolic pH to varying degrees when expressed from cDNA, only M2 and MCM could at least partially complement an M2-null virus in a trans-complementation system. This data suggests that while the CM2 ion channel activity is similar to that of M2, sequences in the ectodomain and/or cytoplasmic tail play important roles in infectious virus production. This thesis suggests that the structure of the membrane proximal region of the M2 cytoplasmic tail may stabilize the membrane distal region, which mediates genome incorporation.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7RX993V

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