Through co-evolution herpesviruses have acquired selective strategies to exploit their hosts, effectively allowing life-long persistence in the host. Tumor-associated herpesviruses, also known as the gamma subfamily of herpesviruses, are distinct in their ability to establish latent infections in lymphocytes and cause benign or malignant tumors in the infected hosts. The two human gammaherpesviruses are Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Given the critical role of type-I interferons (IFN α/β) in the host defense against viruses, it is not surprising that herpesviruses have encoded proteins to counteract the type-I IFN response. Our previous work employing the rodent gammaherpesvirus, murine gammaherpesvirus-68 (MHV-68) determined that a protein encoded by open reading frame (ORF) 54 counteracts the type-I IFN response, which aids the efficient establishment of lifelong persistent infection in the host (Leang, R.S., et al., 2011). In this dissertation, we elucidated the molecular mechanism by which the homolog protein encoded by KSHV's ORF54 antagonizes type-I IFNs and its biological significance in the context of infection. My thesis research led to two significant findings:
1. We identified a novel viral antagonistic mechanism of IFNs through targeting the endosomal trafficking of the interferon / receptor subunit 1 (IFNAR1).
2. We provide evidence to the hypothesis that this mechanism is evolutionarily conserved among all herpesviruses.
Explicitly we established lysosomal degradation mediated by the endosomal sorting complexes required for transport (ESCRT) machinery as the cellular pathway hijacked by ORF54 to down-regulate IFNAR1. Of note to this pathway is the necessary role of Ubiquitin Associated Protein 1 (UBAP1), a subunit of the ESCRT-I complex, for ORF54 to shuttle endocytosed IFNAR1 towards lysosomes. Moreover, we showed that ORF54 does not induce cellular signals that are known to promote IFNAR1. Indeed, ORF54 does not affect IFNAR1 internalization. This feature distinguishes ORF54 from other known viral mechanisms that induce IFNAR1 degradation.
Our data indicate that ORF54 interacts with UBAP1 as well as with IFNAR1, serving as an adaptor to recruit the endocytosed receptor protein to the ESCRT machinery. Ubiquitination of endocytosed proteins is a critical sorting signal for ESCRT. Because ORF54 can bridge the interaction between IFNAR1 and UBAP1 without IFNAR1 being ubiquitinated, it facilitates the introduction of any endocytosed IFNAR1, ubiquitinated or not, into the ESCRT-mediated lysosomal degradation pathway. Furthermore, we showed that another cell surface protein, epidermal growth factor receptor (EGFR), while also undergoing constitutive endocytosis as IFNAR1, is not targeted by ORF54. And yet, IFNAR1 is not the only cell surface receptor targeted by ORF54; we found that gp130 can be down-regulated as well. Therefore, we conclude that ORF54 can selectively target endocytosed surface proteins and bypass vital regulatory steps that mark proteins for degradation.
ORF54 is a functional 2’-deoxyuridine 5’-triphosphate pyrophosphatase (dUTPase) and we previously showed that this enzymatic activity is not required for ORF54 to down-regulate IFNAR1. All herpesviruses, alpha-, beta-, and gamma subfamilies, encode orthologs dUTPases. However, the dUTPase of betaherpesvirus does not have nucleotide hydrolyzing activity. We demonstrated that dUTPases of all three subfamilies interact with UBAP1 and down-regulate IFNAR1. My thesis work has uncovered an evolutionarily conserved viral mechanism antagonizing the innate immune response, suggesting a novel, critical role of herpesviral dUTPases for the establishment of persistent infection and viral pathogenesis.