A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress

Abstract

Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes.

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress

“…Capsids of alphaherpesviruses move from the NRC to the nuclear rim by diffusion rather than by dependence on F actins (73)(74)(75). However, the migration of HCMV capsids is dependent on F actins (13,75). Although the two subunits of NEC are highly conserved across the Herpesviridae family (pUL50 and pUL53 of HCMV, pUL34 and pUL31 of HSV-1 and pseudorabies virus, ORF27 and ORF29 of varicella-zoster virus, and BFRF1 and BFLF2 of Epstein-Barr virus [75]), viruses egress in different ways.…”

“…HCMV capsids have a diameter of about 85 nm, which prevents their direct transport into the cytoplasm through intact nuclear pores (which have a diameter of about 39 nm) (6,(10)(11)(12). Therefore, HCMV nuclear egress occurs in several steps: (i) capsids move from NRCs toward the periphery of the nucleus via F-actin filaments (13), which may help capsids gain contact with the inner nuclear membrane (INM). (ii) Viral nuclear egress complexes (NEC), encompassing viral proteins, such as pUL50, pUL53, and RASCAL, recruit viral kinase pUL97 and cellular proteins, including p32/ gC1qR, emerin, protein kinase C, etc., to phosphorylate nuclear lamins.…”

Getting to and through the inner nuclear membrane during herpesvirus nuclear egress

“…The first step, capsid movement to the nuclear periphery has recently become controversial, with some studies suggesting roles for the nuclear actomyosin system in capsid motility [16,17,22 •• ], and others not [18,19 •• ]. Additional studies are needed to rigorously test the capacity of different herpesvirus capsids to undergo directed movement in the nucleus.…”

“…More recently, however, whether herpesvirus infection induces nuclear F-actin, and whether capsids undergo actin-dependent, directed movement toward the nuclear periphery have become controversial [18, 19 •• , 20, 21 22 •• ]. One report found that infections with several herpesviruses did not induce nuclear F-actin in murine fibroblasts stably expressing the actin-binding peptide, LifeAct-GFP [18].…”

“…In contrast to some of these findings, it was shown that HCMV, but not HSV-1 infection, induces nuclear F-actin in human fibroblasts stably expressing a nuclear-localized version of LifeAct-GFP [22 •• ], and that LatA could fully depolymerize these filaments. Furthermore, electron microscopy revealed that LatA treatment caused a significant decrease in nuclear egress (number of cytoplasmic capsids) and in the percentage of capsids that had moved out of the nuclear interior towards the periphery.…”