MECHANISM AND STAGES OF PACKAGING OF VP8, THE MAJOR TEGUMENT PROTEIN OF BOVINE HERPESVIRUS-1

VP8 (pUL47), the major tegument protein of bovine herpesvirus -1 (BoHV-1), is crucial for viral replication and induction of host immune responses. VP8 (pUL47) translocation from the nucleus to the cytoplasm and subsequently to the Golgi results from its phosphorylation within the nucleus by pUS3. VP8 (pUL47) phosphorylation mutant contains a significantly lower amount of VP8 (pUL47) (~30%) than wild type virus. Outside the context of infection, VP8 (pUL47) is translocated to the cytoplasm if co-transfected with pUS3 encoding plasmid, but remains cytoplasmic and is not translocated to the Golgi. Based on these previous studies, we hypothesized that VP8 (pUL47) is partially packaged in the perinuclear region, and localisation of VP8 at the Golgi for final packaging involves another viral factor, presumably a glycoprotein. Mass spectrometry studies indicated presence of VP8 (pUL47), and another tegument protein, VP22 (pUL49), in the perinuclear and mature virus particles. Co-immunoprecipitation and confocal microscopy confirmed an interaction between VP8 (pUL47) and VP22 (pUL49) and their co-localisation in the perinuclear region, respectively. In cells infected with virus lacking the VP22 (pUL49)-encoding gene, VP8 (pUL47) was absent from the perinuclear space, and the amount of VP8 (pUL47) in the purified mature virus was reduced by approximately 33%. To identify the viral factor(s) responsible for the localisation of cytoplasmic VP8 (pUL47) at the Golgi, a screening of co-precipitating glycoproteins was performed, and glycoprotein M (gM) was observed to be an interaction partner of VP8 (pUL47) during infection, as well as outside the context of infection. VP8 (pUL47) and gM (pUL10) co-localised at the Golgi in infected cells, and gM (pUL10) was sufficient for localisation of VP8 (pUL47) at the Golgi outside the context of infection. In recombinant virus lacking gene encoding gM (ΔgM- BoHV-1), the localisation of VP8 (pUL47) at the Golgi was impeded, and restored with the restoration of gM (pUL10). Analysis of purified mature virus from ΔgM- BoHV-1 infected cells indicated a reduction of approximately 65% in the amount of VP8 (pUL47). The results of this research add to the knowledge of the stages and proteins involved in the assembly of the tegument layer of BoHV-1 with focus on the major tegument protein, VP8 (pUL47).

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Microbiology and Immunology

Grantor

University of Saskatchewan

Year dc:date.issued

2023

Author dc:creator

• Sucharita, Soumya

Advisor dc:contributor.advisor

• van den Hurk, Sylvia

Committee members dc:contributor.committeemember

• Chelico, Linda
• Wilson, Joyce
• Falzarano, Darryl
• Zhou, Yan
• Chowdhury, Shafiqul

• BoHV-1, VP8

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10388/14541

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/14541