Date of Award

12-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Molecular Sciences

Research Advisor

Michael A. Whitt, Ph.D.

Committee

Robert Belland, Ph.D. John Cox, Ph.D. Lawrence Pfeffer, Ph.D. James Patrick Ryan, Ph.D.

Abstract

Vesicular Stomatitis Virus (VSV) is a simple, enveloped, nonsegmented negative-strand RNA virus and is the prototype rhabdovirus to study viral entry, transcription, replication, and assembly. The matrix protein (M) of VSV is a central component of the viral replication cycle. While being the smallest of the viral proteins it is multifunctional and is involved in uncoating, cytopathic effects (CPE), and assembly of the virus. M protein interactions involved in the uncoating and assembly of VSV have been examined in this dissertation.

Uncoating of VSV involves dissociation of M from the ribonucleoprotein core (RNPs) of the virus. Current models of VSV uncoating propose that following membrane fusion M protein is released from the RNP with subsequent diffusion of M into the cytoplasm and distribution of some of the released M to the nucleus of a host cell. The studies in Chapter 2 investigated where in the endocytic pathway uncoating occurs, where M is located following uncoating, and the role of the cytoskeleton in distribution of input M by using a VSV, containing fluorescent M protein (rVSV-M-Lumio-Green). I found that uncoating occurs primarily in early endosomes and results in the majority of M remaining associated with the endosomal membrane which eventually localizes to the perinuclear recycling endosomes. A small fraction of M, which is presumably released into the cytosol, gets delivered to the nuclear envelope, and I found that the typical polymerized actin or microtubules within host cells were not required for distribution of M to the nuclear envelope.

Uncoating and assembly of the VSV genome occurs on membranes within the cytoplasm of the host cell. Exactly how both of these processes can occur in the same environment (e.g. the cytoplasmically exposed membrane surface) without modification of M protein by phosphorylation, cleavage, or some other change has been an intriguing question in the field. In Chapter 3 I present results showing a pH effect on the M-Lumio-Green protein fluorescence in vitro and during the endocytosis of virions which was dependent on G protein. I also observed that low pH enhanced the release of M from rVSV-wt virions, which suggested that acidification of the virus interior results in the dissociation of M contacts within the virus enhancing the uncoating process.

An exposed protease-sensitive loop located between amino acids 120 to 129 of M has been shown to be important for M protein self-association and has been proposed to be crucial for assembly of virions. This knowledge comes from protease treated, purified M protein and not from mutagenesis studies. In Chapter 4 I examined mutations in the exposed loop and in particular a conserved aspartate at residue 125 of a conserved LXD sequence. I found that virions with mutations at residue 123 or 125 of the LXD motif have two phenotypes; 1) an assembly defect and 2) reduced viral protein synthesis starting at 4 hours post infection. These two phenotypes have not been separated genetically and the LXD motif may represent a motif of M involved in assembly and support of VSV protein translation.Vesicular Stomatitis Virus (VSV) is a simple, enveloped, nonsegmented negative-strand RNA virus and is the prototype rhabdovirus to study viral entry, transcription, replication, and assembly. The matrix protein (M) of VSV is a central component of the viral replication cycle. While being the smallest of the viral proteins it is multifunctional and is involved in uncoating, cytopathic effects (CPE), and assembly of the virus. M protein interactions involved in the uncoating and assembly of VSV have been examined in this dissertation.

DOI

10.21007/etd.cghs.2008.0213

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Viruses Commons

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