KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS REMODELS THE NUCLEUS AND ENDOPLASMIC RETICULUM DURING INFECTION

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma, multicentric Castleman’s disease and KSHV-induced cytokine syndrome. KSHV follows a biphasic infectious cycle: upon infection it establishes latency with the genome maintained as an episome tethered to host chromatin, and cellular stress triggers expression of the replication and transcription activator RTA (ORF50) which initiates lytic replication, a program featuring heightened viral gene expression and genome replication. Lytic replication places a heavy burden on the endoplasmic reticulum (ER), the site of folding and modification of secreted and transmembrane proteins and activates the unfolded protein response (UPR). The UPR is coordinated by three ER-localized sensors, PERK, IRE1 and ATF6, which initiate complementary transcriptional programs that expand folding capacity and lipid synthesis, while PERK signaling attenuates cap-dependent translation and can trigger apoptosis if stress is unresolved. KSHV inhibits each arm of the UPR during lytic replication, but the viral factors and mechanisms responsible are incompletely understood. In this thesis I report that the early viral E3 ubiquitin ligase K3 directs K63-linked polyubiquitination of PERK, targeting PERK for lysosomal degradation and thereby terminating PERK signaling. ER stress enhanced K3-dependent PERK turnover, consistent with signal-dependent PERK degradation which converts PERK activation into PERK removal. I demonstrate that the viral G protein-coupled receptor vGPCR induces ER stress and potentiates K3-dependent PERK degradation, and that K3 reciprocally ubiquitinates vGPCR to promote its lysosomal clearance. Therefore, K3 regulates both a source of ER stress and the principal PERK-dependent response. Infection with a K3-deficient virus results in the dysregulated accumulation of viral proteins despite unchanged transcription, implicating K3 in post-transcriptional control of viral proteostasis. Subsequent ultrastructural and live-cell microscopy revealed extensive nuclear membrane remodeling during lytic replication in both WT and ∆K3 infections, pertaining to expansion of the Type-I nucleoplasmic reticulum. These invaginations serve as sites for primary envelopment and nuclear egress, and I document genetically and chemically labeled capsids traffic through these compartments into the cytosol. Together, these studies show that KSHV coordinates robust remodeling of ER and nuclear membranes to support productive infection, with K3 central to manage ER-stress and viral protein proteostasis.