Characterising stress granule formation in bat and porcine cells to understand stress signalling regulation by SADS-CoV and HKU2 accessory proteins

Coronaviruses can infect a wide range of species causing significant disease and economic losses. They arecapable of crossing the species barrier and infecting a new host, often resulting in a highly pathogenic disease.Since 2017, there have been 5 reported outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) in China causing a fatal porcine enteric disease. Phylogenetic analysis has shown coronavirusinterspecies transmission events have ancestral origins in bats, in particular SADS-CoV has high sequencehomology to bat-CoV Hong Kong University 2 (HKU2) which is epizootic to Chinese horseshoe bats.The major differences between HKU2 and SADS-CoV lie in the accessory proteins. Between HKU2 andSADS-CoV there is gain of a new ORF 7b. Between these viruses are four strains of SADS related (SADSr)-CoVs from bats with intermediate greater sequence homology to SADS- CoV. It is possible that the ORF 7bsequence contributes to SADS-CoVs ability to adapt to specific hosts and alter stress signalling responses.Accessory proteins have no homology in number or function between coronaviruses, these proteins are notrequired for replication but are expressed throughout infection and are fundamental in inhibiting anti-viralpathways. Despite this, the role of coronavirus accessory proteins in stress signalling is understudied and inenabling host switching is not known. Notably, viruses from diverse families have been found to modulatestress granule (SG) formation in infected cells by associating with important SG effector proteins. SGs areknown to sequester viral mRNA preventing translation and participate in pathogen sensing, innate immunity,and translational control. Coronavirus accessory proteins are known to play a role in regulating stresssignalling. Interestingly, differences in signalling pathways between species can impact the ability of a virus toregulate anti-viral responses.As there is huge potential for future transmission of novel coronaviruses from bats into new host species, it isimportant to understand the ability of SADS-CoV accessory proteins in regulating stress signalling pathwaysin bats and pigs. In this study the interactions between SADS-CoV and HKU2 (including SADSr-CoVintermediate viruses) accessory proteins and stress signalling, specifically characterising virus-inducedmodulations in SG abundance and how this may play a fundamental role in dictating the success of viralreplication will be explored in bat and porcine cells. This may have wider applications to understanding theviral changes that are necessary to cross the species barrier in future.This work determines that in bat and porcine cells transfected with SADS-CoV and HKU2 accessory proteinsthe proteins localise in majority to the endoplasmic reticulum. Multiple bat and porcine cell lines can respondto different stressors mounting G3BP1-positive stress granules. Amongst bat and porcine cells similaritieswere observed in SADS-CoV 3a and HKU2 3 and SADS-CoV 7a and HKU2 7 stress granules regardless ofthe type of stress. Overall, in bat cells the stress response was more inhibited compared to porcine cells thatmounted a stress response with more stress granules and larger stress granules. Notable differences betweenthe viruses lie with SADS-CoV 7b and may hold answers to increased immune evasion, interferon antagonism,inflammation induction and apoptosis modulation between SADS-CoV and closely related bat-CoV HKU2.