Date of Award

2017

Embargo Period

8-1-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Paula Traktman

Second Advisor

David Long

Third Advisor

Ashley Cowart

Fourth Advisor

Cynthia Wright

Abstract

Vaccinia is the model virus used to study the lifecycle of poxviruses, large DNA viruses that include agents of human disease such as smallpox and monkeypox. Unusual for DNA viruses, poxviruses replicate in the cytoplasm of the cell, autonomous from the nucleus. Because of this autonomy, the virus encodes a large number of genes devoted to replication and morphogenesis. The initial steps of morphogenesis, known as membrane biogenesis, remain unclear. The viral membrane is thought to originate from the endoplasmic reticulum; however, how the membrane is diverted from that organelle is currently not known. Various viral proteins have been identified as being vital to this process. We have been developing genetic tools to study the A30.5 and L2 proteins, two of the regulatory proteins that reside in the ER membrane. Although these proteins are thought to be essential for membrane biogenesis, their mode of action remains unknown. In order to dissect their mode of action, identify functional domains, and investigate protein-protein interactions, we have generated an L2 complementing cell line (CV1:L2HA) and an L2 deletion virus (vΔL2) as well as two inducible A30.5 viruses (vind3XFA30.5; vindA30.5V5). With these reagents, we will assess the full range of phenotypes observed when these proteins are absent. vΔL2 is severely compromised in two different non-complementing cell lines. For structure/function analysis, we will perform complementation analyses with a variety of mutated alleles to identify key domains within L2. The Tet inducible A30.5 viruses, in contrast, are only modestly impaired, with viral yield reduced 3-16 fold in the absence of inducer. The inducible viruses are somewhat leaky, with 2-11% of A30.5 expression under non-induced conditions. To determine whether this leakiness is the cause of the weak phenotype of the vindA30.5 viruses, we are now generating a complementing CV1:3XFLAGA30.5 cell line that will be used to generate a vΔA30.5 deletion virus. We also investigated some structural aspects of A30.5 and L2, including covalent dimerization and phosphorylation status. From our study, neither protein appears to form a covalent dimer. We concluded that A30.5 is phosphorylated.

Rights

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