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

Abstract Herpesvirus egress begins with primary envelopment of newly assembled capsids at the inner nuclear membrane (INM) facilitated by a viral nuclear egress complex (NEC). Primary envelopment has been observed at the peripheral INM as well as at structures known as nuclear infoldings. Nuclear infoldings resulting from invaginations of the INM are known as Type-I nucleoplasmic reticulum (NR), whereas infoldings of both INM and outer nuclear membrane (ONM) are known as 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 invaginations, but primary envelopment was restricted to Type-I NR and the peripheral INM. Over a time-course of infection, DNA-containing KSHV C-capsids were frequently observed budding into nuclear infoldings contiguous with the INM and sparsely decorated with nuclear lamina, characteristic of 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, and drives de novo membrane biogenesis and membrane curvature required for NR expansion; this may provide sufficient Type-I NR to match requirements for KSHV nuclear egress. The selective utilization of the Type-I NR for primary envelopment despite the concurrent expansion of the Type-II NR suggests a mechanism to target C-capsids into the Type-I NR. We also observed accumulation of de-enveloped KSHV C-capsids in 2 nd order nuclear infoldings, suggesting that primary envelopment and de-envelopment can occur not only at the nuclear periphery, but also at the Type-I NR. Importance Herpesvirus capsids assemble in the cell nucleus but are too large to exit via nuclear pores. Instead, they invade the inner nuclear membrane and acquire a provisional lipid envelope that is quickly shed through fusion with the outer nuclear membrane, a neat trick that delivers the capsid to the cytoplasm for subsequent steps in assembly and egress. Dynamic rearrangements of the nuclear membrane include invaginations that plumb the depths of the nucleus to facilitate cargo trafficking. Here, we demonstrate that during Kaposi’s sarcoma-associated herpesvirus (KSHV) replication, nuclear membrane infolding and expansion events increase, coinciding with recruitment of a host enzyme required for phosphatidyl choline synthesis to these sites. We observed an abundance of KSHV capsids at infoldings of the inner nuclear membrane, as well as free capsids in the interior of second order infoldings suggestive of de-envelopment events occurring prior to infold retraction. This complements a more well-established mechanism of KSHV egress at the nuclear periphery, and indeed, we observed concomitant egress of KSHV capsids via both mechanisms from the same nucleus. Similar observations for other herpesviruses by other groups suggests that this versatility in nuclear egress mechanisms is conserved.

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