Auxin-inducible Degron (AID) to Dissect Kaposi's Sarcoma associated Herpesvirus (KSHV) LANA protein function

Protein knock-down with an inducible degradation system is a powerful tool to study proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposis Sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-AID (mAID) tag at the LANA N-terminus with KSHV BAC16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-tagged LANA. Incubation with 5-phenyl-indole-3-acetic acid (5-Ph-IAA), a derivative of natural auxin, rapidly degraded LANA protein within 1.5 hours. In contrast to our hypothesis, depletion of LANA not only failed to trigger lytic reactivation but rather decreased inducible lytic gene expression when we triggered reactivation with a combination of ORF50 protein expression and sodium butyrate treatment. Decreased overall lytic gene induction seemed to associate with a rapid loss of KSHV genomes in the absence of LANA. Furthermore, we found that small cell fractions harbor non-depletable LANA dots in the presence of 5-Ph-IAA. In the cell population containing degradation-resistant LANA, induction of lytic reactivation was strongly attenuated. These results suggest that (i) there are at least two populations of LANA dots in cells, (ii) local nuclear environment and its epigenetic effects on the episomes are heritable to daughter cells; this biological had substantial effects in degree of KSHV reactivation, and finally (iii) LANA may have an additional function in protecting KSHV episomes from degradation. IMPORTANCEKSHV LANA protein plays a wide variety of roles in latency maintenance and lytic gene expression. We adapted the inducible protein knockdown approach to examine its role directly, and revealed that there are cell populations that possess viral episomes insensitive to reactivation stimuli. Viral reactivation is known to be highly heterogenic, and our observations suggest that LANA tethering sites on host chromatin may play a critical role in determining diverse responsiveness to the stimuli. We also demonstrated that depletion of LANA leads to rapid reduction of viral genome, which suggests that LANA might be actively protecting latent viral genome from degradation. These results add novel insights into the role of LANA in latency maintenance and regulation of lytic reactivation.