Comparative analysis of ISGylation during the antiviral response against Paramyxovirus infection
Creators: name: Seaton, Andrew Vasili
Publisher: name: The University of St Andrews
Year: 2024
Subjects: ISG15
ISGylation
Paramyxovirus
Interferon response
Interferon stimulated gene

description: Viral infection induces a significant antiviral response through the interferon response. This response leads to the production of hundreds of interferon stimulated genes (ISGs) including ISG15; a Ubl protein with multiple roles in the antiviral response. Within the antiviral response ISG15 plays a role as a negative regulator of the IFN response with loss of ISG15 generating an overactive response, plays a role in extracellular signalling and is capable of post-translationally modifying host and viral proteins through a process known as ISGylation. ISGylation is known to alter functions of proteins through aiding host defence and playing an antiviral role when modifying viral proteins. We have identified a novel modification of parainfluenza virus type-5 (PIV5) phosphoprotein (P protein) and through use of CRISPR/Cas9 and immunoblot analysis have confirmed that this modification is ISG15. Further to this we have shown that this appears to be the only modified protein of PIV5. As the P protein plays a critical role in viral replication we took a comparative approach through analysis of effects of ISGylation PIV5, PIV2 and PIV3. In order to comparatively analyse effects of ISGylation on plaque size we used a computationally-aided plaque assay approach to determine effects of ISGylation. Our initial dataset suggested that ISGylation had little to no effect on replication of these paramyxoviruses. However further investigation using IFIT1.ko cell lines yielded data suggesting that loss of ISGylation lead to an increase in plaque size in PIV2 and PIV5 suggesting ISGylation was antiviral to these viruses but noted no effect on PIV3. Importantly we noted that loss of ISG15 lead to a decrease in plaque size which is attributed to the role of ISG15 as a negative regulator of the antiviral response. These findings suggested that ISGylation plays a minor antiviral role with respect to PIV2 and PIV5 and potentially may impact other members of the Paramyxoviridae family, however this requires further investigation. As noted, we observed small plaque sizes present within ISG15.ko IFIT1.ko cells, particularly those infected with PIV3. We further investigated these plaques and observed a strange phenotype wherein immunostaining of viral plaques yielded only tiny viral plaques while counter staining for cell viability revealed large areas of cell death surrounding these small areas of viral activity. Further investigation into this suggested an IFN-alpha signalling dependent pathway causing cell death highlighting differences between the IFN-alpha and beta responses. Overall our data has shown that ISGylation is a minor antiviral factor for the Paramyxoviridae family, especially in comparison to the major antiviral role ISG15 plays within its negative regulation of the IFN response. This major role of ISG15 as a negative regulator is detracted from as cells lacking ISG15 undergo high levels of cell death in response to signalling from infected cells. Further investigations would be needed to identify the antiviral mechanism of ISGylation as well as whether other Paramyxoviridae are affected by this modification as well as the exact pathway that triggers cell death within ISG15.ko cells.