Role of Herpes Simplex Virus 1 Immediate Early Protein ICP22 in Viral Nuclear Egress

Abstract

In order to investigate the novel function(s) of the herpes simplex virus 1 (HSV-1) immediate early protein ICP22, we screened for ICP22-binding proteins in HSV-1-infected cells. Our results were as follows. (i) Tandem affinity purification of ICP22 from infected cells, coupled with mass spectrometry-based proteomics and subsequent analyses, demonstrates that ICP22 forms a complex(es) with the HSV-1 proteins UL31, UL34, UL47 (or VP13/14), and/or Us3. All these proteins were previously reported to be important for viral egress through the nuclear membrane. (ii) ICP22 colocalizes with UL31 and UL34 at the nuclear membrane in wild-type HSV-1-infected cells. (iii) The UL31-null mutation prevents the targeting of ICP22 to the nuclear membrane. (iv) The ICP22-null mutation resulted in UL31 and UL34 being mislocalized in the endoplasmic reticulum (in addition to the nuclear membrane) and significantly reduced numbers of primary enveloped virions in the perinuclear space, although capsids accumulated in the nuclei. Collectively, these results suggest that (i) ICP22 interacts with HSV-1 regulators of nuclear egress, including UL31, UL34, UL47, and Us3 in HSV-1-infected cells; (ii) UL31 mediates the recruitment and anchorage of ICP22 at the nuclear membrane; and (iii) ICP22 plays a regulatory role in HSV-1 primary envelopment, probably by interacting with and regulating UL31 and UL34. Here we report a previously unknown function for ICP22 in the regulation of HSV-1 nuclear egress. IMPORTANCEThe herpes simplex virus 1 (HSV-1) immediate early protein ICP22 is recognized primarily as a regulator of viral gene expression. In this study, we show that ICP22 interacts with the HSV-1 proteins UL31 and UL34, which play crucial roles at the nuclear membrane in HSV-1 primary envelopment during viral nuclear egress. We also demonstrate that UL31 is required for the targeting of ICP22 to the nuclear membrane and that ICP22 is required for the correct localization of UL31 and/or UL34. Furthermore, we confirm that ICP22 is required for efficient HSV-1 primary envelopment during viral nuclear egress. Thus, we report, for the first time, that ICP22 plays a regulatory role in HSV-1 nuclear egress. Herpes simplex virus 1 (HSV-1) is one of the best-studied members of the Herpesviridae family, causing a variety of diseases such as mucocutaneous diseases, keratitis, skin diseases, and life-threatening encephalitis (1). After HSV-1 entry into the host cell, the nucleocapsid is transported to a nuclear pore, enabling the entry of the viral genome into the nucleus and initiation of viral gene expression (1). HSV-1 encodes Ͼ80 different proteins that are expressed in a regulated cascade and fall into three major classes, designated immediate early (IE), early (E), and late (L) (1). Viral DNA replication, capsid assembly, and packaging of replicated viral genomes into capsids take place in the nucleus (1). The nascent progeny nucleocapsids of HSV-1, like those of other herpesviruses, then traverse the inner nuclear membrane (INM) and outer ...

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

“…Us3 has been reported to regulate localization of UL31, UL34, UL47 and ICP22 at the nuclear rim in HSV-1-infected cells (3,7,8). In the absence of Us3 protein or its catalytic activity, these viral proteins were shown to accumulate aberrantly in punctate structures at the nuclear rim in HSV-1-infected cells (3,7,8).…”

“…We next investigated whether p32 interacted with components of the HSV-1 NEC other than UL47, including UL31, UL34, Us3, and ICP22, all of which have been reported to be important for viral nuclear egress at the nuclear rim (4,7,8,57), and performed two series of experiments. In the first series of experiments, Vero cells were infected with wild-type HSV-1(F), YK536 (MEF-UL47), YK538 (MEF-UL34) encoding MEF-tagged UL34, or YK539 (MEF-UL31) encoding MEF-tagged UL31 (Fig.…”

“…Commercial rabbit polyclonal antibody against VP23 (CAC-CT-HSV-UL18; CosmoBio) and commercial mouse monoclonal antibodies against Flag (M2; Sigma), Myc (PL14; MBL), ICP8 (10A3; Chemicon), VP5 (3B6; Virusys), and ␤-actin (AC15; Sigma) were used in this study. Rabbit polyclonal antibody to UL34, UL31, ICP22, Us3 and UL47, mouse polyclonal antibody to UL31, and chicken polyclonal antibody to UL34 (a generous gift from R. Roller) were described previously (3,7,8,50,56). Rabbit polyclonal antibody to UL34 was used for immunoblotting, and chicken polyclonal antibody to UL34 was used for immunofluorescence.…”

“…A heterodimeric complex of herpes simplex virus 1 (HSV-1) proteins UL31 and UL34, which are conserved in all known herpesviruses, is critical for HSV-1 primary envelopment during viral nuclear egress and has been designated the nuclear egress complex (NEC) (1-6). Recently, the HSV-1 NEC has been reported to form a complex with the HSV-1 serine/threonine protein kinase Us3, major HSV-1 structural protein UL47 (also designated VP13/14), and HSV-1 regulatory protein ICP22 (7,8). Among these recently identified components of the HSV-1 NEC, UL47 and ICP22 have been shown to be important for HSV-1 primary envelopment, based on the observations that a UL47-null or ICP22-null mutation significantly reduced the number of primary enveloped virions in the perinuclear space and induced accumulation of capsids in the nucleus (7,8).…”

“…Recently, the HSV-1 NEC has been reported to form a complex with the HSV-1 serine/threonine protein kinase Us3, major HSV-1 structural protein UL47 (also designated VP13/14), and HSV-1 regulatory protein ICP22 (7,8). Among these recently identified components of the HSV-1 NEC, UL47 and ICP22 have been shown to be important for HSV-1 primary envelopment, based on the observations that a UL47-null or ICP22-null mutation significantly reduced the number of primary enveloped virions in the perinuclear space and induced accumulation of capsids in the nucleus (7,8). In contrast, Us3 has been reported to play an important role in de-envelopment of HSV-1 nucleocapsids.…”