Investigating the role of the West Nile virus and Koutango virus envelope protein in virulence phenotypes using structural protein chimeras

Flaviviruses have caused, and continue to cause, both sporadic and regular outbreaks globally which result in significant global disease burden. To better understand how different portions of the virus contribute to transmission and disease caused by these viruses, researchers have found ways to isolate viral components. While methods such as site-directed mutagenesis, virus-like particles, and trans-complementation have been useful, they all fall short of being able to study the contribution of an entire protein in the context of a replicative virus. The generation of chimeric flaviviruses bridges this gap by being able to study the contributions of a single protein or domain in the context of a replicative virus. Previous work by McAuley et al. 2016 identified the unexpectedly attenuated phenotype of the West Nile virus/Koutango virus envelope protein domain III chimera (WNV/KOUV-EIII), which was generated from the combination of two highly virulent viruses. It was hypothesized that attenuation was due to intra- and/or inter-molecular interactions between the WNV E protein domains important for efficient viral replication and pathogenesis being disrupted by chimerization in WNV/KOUV-EIII. To test this hypothesis, two additional chimeras (WNV/KOUV-prME and WNV/KOUV-E) and three WNV/KOUV-EIII mutants were generated to identify critical determinants of the attenuation. Following a series of biophysical, in vitro and in vivo assessments, it was determined that the attenuation of the WNV/KOUV-EIII chimera was mainly due to the 370I in the KOUV-EIII in the contact of WNV-EI/EII, which is associated with increased pH sensitivity but independent of thermal stability. In addition to the experiments described above, investigation into KOUV through the generation of an infectious clone (ic) enabled further study of the WNV/KOUV E proteins. Using the newly created KOUV ic, KOUV/WNV-EIII and -prME chimeras were produced and characterized. Unfortunately, the inclusion of an unexpected E protein mutation made interpretation of the results challenging. Further study of the WNV E protein was done following the discovery of a 93nt insertion into the E protein during routine site-directed mutagenesis. These experiments highlighted the potential for and tolerance of manipulation by insertion for the WNV E protein. Overall, the works described in this dissertation highlight the complex roles of the E protein with regard to pathogenicity, multiplication rates, and biophysical stability. Using a series of chimeras and mutants, several attenuating mutations and in vitro phenotypes have been found to be associated with a decrease in mouse virulence, mediated by the KOUV EIII.

Name thesis:degree_name

Microbiology and Immunology (Doctoral)

Discipline thesis:degree_discipline

Microbiology

Grantor

The University of Texas Medical Branch at Galveston

Year dc:date.issued

2023

Author dc:creator

• Haila, Gabriel 1996-

Advisor dc:contributor.advisor

• David Beasley

Language dc:language.iso

English

Handle dc:identifier.uri

https://hdl.handle.net/2152.3/12850

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/12850