Date of Award
12-2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biology
First Advisor
Dr. Margo Brinton
Second Advisor
Dr. Richard Plemper
Third Advisor
Dr. Markus Germann
Abstract
Simian hemorrhagic fever virus (SHFV) is an enveloped, single-stranded, positive sense RNA virus that infects monkeys and has the largest genome (15.7 kb) in the family Arteriviridae. The genome 5′ end encodes two polyproteins that cleaved into 14 individual nonstructural proteins (nsps) by five viral proteases. There are three nsp1 (nsp1α, nsp1β, and nsp1γ) proteins and one nsp2 protein that each contain an active papain-like protease (PLP). The genomes of non-simian arteriviruses encode one or two nsp1 proteins. SHFV infectious clones with the individual nsp1coding regions deleted produced lower levels of sg mRNAs but normal levels of genomic RNA. The SHFV structural proteins are encoded in the 3′ third of the genome and are translated from a nested set of 3′ and 5′ coterminal sg mRNA. The minus strand templates for the sg mRNAs are produced by a discontinuous transcription mechanism that is regulated by transcription-regulating sequences (TRSs) in the genome. Our previous RNAseq data showed that the relative abundance of the individual SHFV 3′ ORF sg mRNAs differ and identified multiple functional TRSs for most of the 3′ ORFs. The SHFV genome RNA secondary structure was predicted by a SHAPE analysis and the locations of the functional TRSs were mapped on these structures. The conserved sequence (5′-AACC-3′) was identified within most functional TRSs. Nonsense mutations were made in SHFV infectious clones to disrupt a TRS RNA structural context (ORF2b and ORF3) or to change the TRS sequence (TRS4′ and TRS7). The TRS sequence mutations had the greatest effect on reducing sg mRNA abundance and virus yield. The genome reverted to its original sequence after passage (structure mutations) or by four days after transfection (TRS sequence mutations). The data suggest that both the RNA structural context and the TRS sequence regulate sg mRNA abundance. LC/MS-MS analysis performed on V5 immunoprecipitated proteins from SHFV-infected cells transfected with nsp1α-V5 mRNA enriched cellular proteins associated with mRNA splicing, mRNA transport, and spliceosomes. Co-immunoprecipitation assays revealed a direct interaction between nsp1α and Gemin5. Genim5 is a component of the survival of motor neurons (SMN) complex responsible for spliceosome assembly in the cytoplasm. An additional LC/MS-MS analysis performed on V5 immunoprecipitated proteins from SHFV-infected cells transfected with nsp1b-V5 mRNA enriched the nuclear proteins TLE1, TLE3 and TLE4 that negatively regulate cellular transcription. The data suggest a role of nsp1b in facilitating inhibition of the transcription of cellular proteins involved the innate response.
Recommended Citation
Sadhwani, Heena, "Functional analysis of simian hemorrhagic fever virus non-structural proteins 1, transcription-regulating sequences and viral RNA structures during viral replication." Dissertation, Georgia State University, 2024.
https://scholarworks.gsu.edu/biology_diss/281
File Upload Confirmation
1