Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that causes Kaposi's sarcoma (KS) and two lymphoproliferative diseases. We previously reported KSHV ORF52/KicGAS (KSHV inhibitor of cGAS) to inhibit cGAS (cyclic GMP-AMP synthase), the principal sensor of cytosolic DNA. Compared to its target protein, cGAS, whose intracellular localization is closely regulated, the predicted molecular weight of ORF52 is relatively small (14 kDa) but remains excluded from the nucleus. As a late-phased lytic protein of KSHV, we also previously reported that ORF52 is required for progeny viral production, likely during virion assembly, a process that takes place in the cytoplasm of host cells. Thus, we speculated its controlled localization to be important for the proper functioning of ORF52. We first performed extensive microscopy and characterized the intracellular localization of KSHV ORF52 in detail. Under different experimental conditions, we observed primarily homogeneous localization of the protein in the cytoplasm, but also saw different patterns of bright focus, perinuclear halo, or wire-loop bundling in a minor population of expressing cells. Furthermore, we confirmed the focus pattern was likely formed by ORF52:DNA condensation, which was due to relocalization of cytoplasmic ORF52 signal induced by cytosolic DNA. Looking into the underlying molecular controls, we located a region near the N-terminus that is critical for the exclusively cytoplasmic localization of ORF52. Further analyses revealed two CRM1-dependent nuclear export signals (NESs), one at the N-terminal domain satisfying a PIK-NES, which is functionally more important. Similar NESs were also identified in other γ-2 (RRV, MHV68, HVS, EHV-2, and BHV-6), but not in γ-1 herpesviruses (EBV, MaHV4). Point mutation at the nuclear-localized EBV ORF52 (BLRF2) at S36 to hydrophobic residues enables relocations to the cytoplasm, suggesting the designated area to be essential for the localization of gammaherpesviral ORF52s, but evolutionarily bifurcated at genus level. Interestingly, even though there are two functional NESs were recognized in it, we noticed that KSHV ORF52 remained cytoplasmic upon treatment of Leptomycin B (LMB), a CRM1 inhibitor. Furthermore, we also observed inconsistencies in response to LMB between KSHV ORF52 and its truncational mutants, as well as among its homologues, suggesting additional controls to the localization that are specific to KSHV. In our attempt to explain this phenomenon, we performed nuclear export reporter assays and mRNA-exporter luciferase reporter assays only to find KSHV ORF52 may inhibit CRM1-mediated nuclear export. Finally, we tested KSHV ORF52 mutants for its inhibition of cGAS-mediated-IFN response, inhibition of CRM1-mediated nuclear export, production of infectious KSHV progeny virion during viral reactivation, and enhancement of microtubule cytoskeleton bundling/polymerization. In each experiment, we observed the key residues for NES that are critical to the cytoplasmic localization to be required. Altogether, these data suggested versatile functions of KSHV ORF52 and the importance of intracellular localization for its normal functioning in the KSHV life cycle, which shed light on future studies on KSVH-associated pathogenesis.
