Role of Host Cell p32 in Herpes Simplex Virus 1 De-Envelopment during Viral Nuclear Egress

Abstract

To clarify the function(s) of the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (also designated VP13/14), we screened cells overexpressing UL47 for UL47-binding cellular proteins. Tandem affinity purification of transiently expressed UL47 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that UL47 interacted with cell protein p32 in HSV-1-infected cells. Unlike in mock-infected cells, p32 accumulated at the nuclear rim in HSV-1-infected cells, and this p32 recruitment to the nuclear rim required UL47. p32 formed a complex(es) with HSV-1 proteins UL31, UL34, Us3, UL47, and/or ICP22 in HSV-1-infected cells. All these HSV-1 proteins were previously reported to be important for HSV-1 nuclear egress, in which nucleocapsids bud through the inner nuclear membrane (primary envelopment) and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Like viral proteins UL31, UL34, Us3, and UL47, p32 was detected in primary enveloped virions. p32 knockdown reduced viral replication and induced membranous invaginations adjacent to the nuclear rim containing primary enveloped virions and aberrant localization of UL31 and UL34 in punctate structures at the nuclear rim. These effects of p32 knockdown were reduced in the absence of UL47. Therefore, the effects of p32 knockdown in HSV-1 nuclear egress were similar to those of the previously reported mutation(s) in HSV-1 regulatory proteins for HSV-1 de-envelopment during viral nuclear egress. Collectively, these results suggested that p32 regulated HSV-1 de-envelopment and replication in a UL47-dependent manner. IMPORTANCEIn this study, we have obtained data suggesting that (i) the HSV-1 major virion structural protein UL47 interacted with host cell protein p32 and mediated the recruitment of p32 to the nuclear rim in HSV-1-infected cells; (ii) p32 was a component of the HSV-1 nuclear egress complex (NEC), whose core components were UL31 and UL34; and (iii) p32 regulated HSV-1 de-envelopment during viral nuclear egress. It has been reported that p32 was a component of human cytomegalovirus NEC and was required for efficient disintegration of nuclear lamina, which has been thought to facilitate HSV-1 primary envelopment during viral nuclear egress. Thus, p32 appeared to be a core component of herpesvirus NECs, like UL31 and UL34 homologs in other herpesviruses, and to play multiple roles in herpesvirus nuclear egress. H erpesvirus nucleocapsids are too large to traverse the nuclear lamina or cross the inner (INM) and outer (ONM) nuclear membranes through nuclear pores. Therefore, herpesviruses appear to have evolved a unique nuclear egress mechanism in which progeny nucleocapsids assembled in the nucleus acquire primary envelopes by budding through the INM into the perinuclear space (primary envelopment), the space between the INM and ONM, and enveloped nucleocapsids then fuse with the ONM to release de-enveloped nucleocapsids into the cytoplasm (de-envelopment) (1, 2). A heterodim...

Characterization of a Herpes Simplex Virus 1 (HSV-1) Chimera in Which the Us3 Protein Kinase Gene Is Replaced with the HSV-2 Us3 Gene

“…In agreement with previous reports (15,21,27,43,74), in Vero cells infected with wild-type HSV-1(F) or YK513 (Us3K220M-repair), UL34 and UL31 proteins colocalized smoothly along the nuclear rim, and in Vero cells infected with YK511 (Us3K220M), UL34 and UL31 colocalized in punctate structures adjacent to the nuclear rim (Fig. 8).…”

“…The NEC has recently been reported to form a complex(es) with HSV-1 UL47, Us3, and ICP22 and cellular proteins p32, CD98 heavy chain, and ␤1 integrin (67,74,80,81), suggesting that the NEC is a high-order complex consisting of these viral and cellular regulatory proteins and probably some not-yet-identified proteins. Among the regulatory proteins, UL47, Us3, and ICP22 are known to be substrates for Us3 (15,37,72,82), and p32 has been reported to contain multiple sites similar to Us3 target consensus sequences (74,83). Therefore, differences in the phosphorylation of HSV-1 and infected-cell regulatory proteins for viral nuclear egress, due to the replacement of HSV-1 Us3 with HSV-2 Us3, may have impaired the function of the highorder NEC, leading to defects in viral nuclear egress.…”

Mapping Murine Corneal Neovascularization and Weight Loss Virulence Determinants in the Herpes Simplex Virus 1 Genome and the Detection of an Epistatic Interaction between the UL and IRS/US Regions

“…The VP11/12, VP13/14, VP22, ICP22, US3, gD, and gI proteins form a virion assembly/egress subnetwork. The VP13/14, ICP22, and US3 proteins work in conjunction with UL31, UL34, and p32 (C1QPB) to form the nuclear export complex (NEC), which promotes virion budding through the nuclear membrane (61,62). VP13/14 contains one vQTLmap-identified SNP (A3S), where the more virulent serine variation forms a casein kinase II (CKII) phosphorylation motif (SXXD/E) (63).…”

Extragenic Suppression of a Mutation in Herpes Simplex Virus 1 UL34 That Affects Lamina Disruption and Nuclear Egress

“…Protein kinases PKC␣ and PKC␦ are recruited to the nuclear periphery upon HSV-1 infection, and they phosphorylate emerin and lamin B (12,13). The cellular protein p32 has also been shown to be recruited to the nuclear periphery during HSV-1 infection, and it interacts with pUL31, pUL34, LBR, and other components of the NEC (14,15).…”