Han DiFollow

Date of Award

5-9-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Margo A. Brinton

Second Advisor

Richard K. Plemper

Third Advisor

Parjit Kaur

Abstract

Simian hemorrhagic fever virus (SHFV) is an enveloped, single stranded, positive sense RNA virus that infects monkeys. SHFV has the largest genome (15.7 kb) in the family Arteriviridae that encodes two polyproteins. These polyproteins are auto-cleaved into nonstructural proteins by three different types of viral proteases. Compared to other arteriviruses, SHFV encodes three, rather than one or two, functional papain-like protease (PLP) 1 domains at the 5′ end of the genome. The catalytic residues and cleavage site(s) were determined for each of the SHFV PLP1 domains. The 3′ end of the SHFV genome encodes 12 structural proteins including a second set of minor structural proteins not found in other arteriviruses. The SHFV structural proteins are translated from a nested set of 3′ and 5′ coterminal subgenomic mRNAs (sg mRNA). Syntheses of the minus strand templates for the sg mRNAs is discontinuous and regulated by transcription regulating sequences (TRSs) in the genome. The SHFV sg mRNAs produced were analyzed by Northern blot assay and 37 additional functional TRSs were discovered in SHFV genome by cloning and sequencing the sg mRNA junction sequences. Next generation sequencing analysis of the SHFV transcriptome confirmed sg mRNA production from all of the discovered TRSs and allowed estimation of the transcription level of each sg mRNA as well as the expression level of each 3′ ORF at early and late times after infection. Some of the sg mRNAs generated from newly discovered TRSs encode new ORFs. Two of these are functionally relevant for efficient virion production in MA104 cells. The potential for constructing a recombinant SHFV expressing a foreign gene from an additional sg mRNA was explored using an SHFV LVR cDNA infectious clone. The position between the two sets of minor structural protein ORFs was found to be tolerant for insertion of a foreign gene. Three different TRSs were inserted individually after the foreign gene and each of them restored the expression of the downstream SHFV structural protein ORF and generated infectious progeny virus. However, only one of these progeny virus that maintained the full-length foreign gene in the genome after three passages.

DOI

https://doi.org/10.57709/8519788

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