Type
Text
Type
Dissertation
Advisor
Wimmer, Eckard | Hearing, Patrick | Krug, Laurie | Carter, Carol.
Date
2015-12-01
Keywords
codon pair bias, vaccine development, Vesicular stomatitis virus | Virology
Department
Department of Molecular Genetics and Microbiology.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/76546
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Codon pair bias (CPB), which has been observed in all organisms, is a neglected genomic phenomenon that substantially affects gene expression. CPB results from synonymous codons that are paired more or less frequently in ORFeomes regardless of codon bias. The effect of an individual codon pair change is usually small, but when amplified by large-scale genome recoding (simultaneously generating hundreds of synonymous mutations without altering codon usage or amino acid sequence), strikingly altered biological phenotypes are observed. Although the molecular mechanism underlying codon pair bias still remains enigmatic, the utility of codon pair bias in the development of live attenuated vaccines was recently demonstrated by recodings of poliovirus (a positive-strand RNA virus) and influenza virus (a negative-strand segmented RNA virus). In an attempt to expand the attenuation spectrum of codon pair bias, I recoded the L and G genes of vesicular stomatitis virus (VSV), a non-segmented negative strand RNA virus. For L gene recoding, I introduced a total of 858 and 623 silent mutations into a 5’-terminal segment of the viral L gene (designated as L1), to create sequences containing either overrepresented (sdmax design) and underrepresented (min design) codon pairs, respectively. These two sequences were designated as L1sdmax and L1min, respectively. Recombinant VSV containing the L1min sequence could not be recovered via a well-established reverse genetics system whereas the introduction of the L1sdmax sequence into the viral genome conferred a modest level of attenuation in cell culture. The phenotype of the L1sdmax virus was unexpected, which might result from a discoordination between translation elongation rate and protein folding kinetics. More strikingly, in mice the L1sdmax virus was almost as immunogenic as the parental strain but highly attenuated. Despite this, the recombinant virus showed a temporarily productive replication in the infected tissues while neither histopathological change nor severe morbidity has ever been found in L1sdmax- infected animals. As for the G gene recoding, I introduced 323 silent mutations into the G gene open- reading frame. The newly generated recombinant virus was designated as Gmin, which contained hundreds of underrepresented codon pairs. Although during the early stages of virus infection significantly less G gene products were produced in infected cells, the Gmin virus replicated just as well as its parental strain in tissue culture and showed neurovirulence in a mouse model. Taken together, my research suggests that L1sdmax, but not L1min or Gmin, is a promising live attenuated vaccine candidate for VSV infection. In addition, these results demonstrate a novel approach to attain a balance between VSV virulence and immunogenicity, which could serve as a paradigm for the attenuation of other negative-strand, non-segmented RNA viruses (NNS viruses). | Codon pair bias (CPB), which has been observed in all organisms, is a neglected genomic phenomenon that substantially affects gene expression. CPB results from synonymous codons that are paired more or less frequently in ORFeomes regardless of codon bias. The effect of an individual codon pair change is usually small, but when amplified by large-scale genome recoding (simultaneously generating hundreds of synonymous mutations without altering codon usage or amino acid sequence), strikingly altered biological phenotypes are observed. Although the molecular mechanism underlying codon pair bias still remains enigmatic, the utility of codon pair bias in the development of live attenuated vaccines was recently demonstrated by recodings of poliovirus (a positive-strand RNA virus) and influenza virus (a negative-strand segmented RNA virus). In an attempt to expand the attenuation spectrum of codon pair bias, I recoded the L and G genes of vesicular stomatitis virus (VSV), a non-segmented negative strand RNA virus. For L gene recoding, I introduced a total of 858 and 623 silent mutations into a 5’-terminal segment of the viral L gene (designated as L1), to create sequences containing either overrepresented (sdmax design) and underrepresented (min design) codon pairs, respectively. These two sequences were designated as L1sdmax and L1min, respectively. Recombinant VSV containing the L1min sequence could not be recovered via a well-established reverse genetics system whereas the introduction of the L1sdmax sequence into the viral genome conferred a modest level of attenuation in cell culture. The phenotype of the L1sdmax virus was unexpected, which might result from a discoordination between translation elongation rate and protein folding kinetics. More strikingly, in mice the L1sdmax virus was almost as immunogenic as the parental strain but highly attenuated. Despite this, the recombinant virus showed a temporarily productive replication in the infected tissues while neither histopathological change nor severe morbidity has ever been found in L1sdmax- infected animals. As for the G gene recoding, I introduced 323 silent mutations into the G gene open- reading frame. The newly generated recombinant virus was designated as Gmin, which contained hundreds of underrepresented codon pairs. Although during the early stages of virus infection significantly less G gene products were produced in infected cells, the Gmin virus replicated just as well as its parental strain in tissue culture and showed neurovirulence in a mouse model. Taken together, my research suggests that L1sdmax, but not L1min or Gmin, is a promising live attenuated vaccine candidate for VSV infection. In addition, these results demonstrate a novel approach to attain a balance between VSV virulence and immunogenicity, which could serve as a paradigm for the attenuation of other negative-strand, non-segmented RNA viruses (NNS viruses). | 135 pages
Recommended Citation
Wang, Bingyin, "Recoding of Vesicular Stomatitis Virus genes by computer-aided design provides a live attenuated vaccine candidate" (2015). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2447.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2447