Investigating Herpesvirus Replication and Virus-Host Interactions During SARS-CoV-2 Co-Infections
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Authors
Payen, Shannon Lynn
Issue Date
2024
Type
Dissertation
Language
Keywords
Coronavirus , Herpesvirus , KSHV , Microbiology , SARS-CoV-2 , Virology
Alternative Title
Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus in the Herpesviridae family. KSHV is an oncogenic virus and is the causative agent of Kaposi's sarcoma (KS), Multicentric Castleman's Disease (MCD), primary effusion lymphoma (PEL), and Kaposi Sarcoma Inflammatory Cytokine Syndrome (KICS). KSHV, like all herpesviruses, has a bi-phasic replication cycle consisting of a latent phase with periodic episodes of lytic reactivation. Within the replication complex, the DNA polymerase processivity factor plays a critical role in viral DNA synthesis through interactions with both viral and cellular components. Previous studies have described various functional domains of ORF59. However, the regions responsible for interactions with viral mRNA transport accumulation protein ORF57 and viral polyadenylated nuclear (PAN) RNA remained unexplored. Using ORF59 deletion mutants in KSHV BACmid, we identified multiple domains of ORF59 (amino acids 51-100, 251-300, and 351-396) that interact with PAN RNA. Notably, the absence of association with ORF59 led to an aberrant localization pattern of PAN RNA, forming aggregates concentrated into foci. Interestingly, amino acids 51-100 were found to be essential for interaction with ORF57, indicating a shared interaction domain for PAN RNA and ORF57. To further elucidate the role of ORF59 in viral replication, a small DsRed-tagged polypeptide spanning amino acids 30-100 of ORF59 was created. Transfection of this polypeptide into iSLK-WT cells resulted in a dominant-negative inhibition of virus replication, leading to decreased infectious virion production. To understand why nuclear punctate formed within specific ORF59 deletion domains, we investigated the role of paraspeckle nuclear bodies, comprising core ribonucleoproteins SFPQ and NONO during lytic KSHV replication. Using SFPQ immunoprecipitation followed by mass spectrometry, we found interactions between SFPQ and KSHV viral proteins, ORF10, ORF59, ORF61, ORF67, ORF11, ORF21, ORF50, ORF52, ORF57, ORF6, ORF25, and ORF69. Further analysis identified the region spanning amino acids 101-150 of ORF59 as the interaction domain with SFPQ. By generating a dominant-negative polypeptide of ORF59 amino acids 101-150, we disrupted the interaction between SFPQ and full-length ORF59, resulting in decreased virus production. Interestingly, co-immunoprecipitation experiments with other human herpesvirus processivity factors revealed similar interaction with SFPQ, suggesting a potentially conserved requirement for SFPQ across multiple herpesviruses. Lastly, with the emergence of SARS-CoV-2 and the ongoing COVID-19 pandemic, the association between human cytomegalovirus (HCMV) seropositivity and increased COVID-19 severity has gained attention. HCMV is a ubiquitous beta-herpesvirus with a high seroprevalence worldwide and is a leading cause of congenital abnormalities and mortality in immunocompromised individuals. Our studies found CD14 + monocytes, one of the primary reservoirs of latency for HCMV, to be susceptible and permissive to SARS-CoV-2 infection. RNA-sequencing analysis revealed significant differences in cytokine-cytokine receptor interactions and inflammatory pathways in HCMV-latent CD14 + monocytes superinfected with replication-competent SARS-CoV-2. These findings provide insights into the heightened inflammatory response associated with co-infection, suggesting potential therapeutic interventions for severe cases of COVID-19 among HCMV-infected individuals. Overall, these studies contribute to our understanding of viral interactions and their implications for disease pathogenesis, highlighting potential therapeutic targets to mitigate the severity of viral infections, particularly in cases of co-infection.
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Creative Commons Attribution 4.0 International