Primary envelopment of Kaposi’s sarcoma-associated herpesvirus at the nucleoplasmic reticulum

Herpesvirus egress begins with primary envelopment of newly assembled capsids at the inner nuclear membrane (INM). Primary envelopment has been observed at the peripheral INM as well as nuclear infoldings. Nuclear infoldings from invaginations of the INM are Type-I nucleoplasmic reticulum (NR), whereas infoldings of both INM and outer nuclear membrane (ONM) are Type-II NR. Here, we report that Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation from latency and lytic cycle progression correlates with increases in both types of NR, but primary envelopment is restricted to peripheral INM and Type-I NR. These Type-I NR structures co-localized with puncta containing CTP:phosphocholine cytidylyltransferase (CCTα), the enzyme that catalyzes the rate-limiting step in phosphatidylcholine (PtdCho) synthesis that drives the de novo membrane biogenesis and membrane curvature required for NR expansion; CCTα recruitment may provide sufficient Type-I NR to facilitate nuclear egress. Despite the concurrent expansion of Type-II NR, primary envelopment involves a mechanism that specifically targets capsids to Type-I NR. Building upon our observation of capsids lacking envelopes in complex higher-order Type-I NR structures, we used polar lipid dyes, CLICK-labeled fluorescent viral genomes, and fluorescent KSHV capsids to track the fate of NR-associated capsids via live cell microscopy. These studies provide evidence for trafficking of NR-associated capsids toward the nuclear periphery and cytoplasm. Taken together, these findings suggest that nuclear egress occurs not only at the nuclear periphery but also at the Type-I NR.