Herpesviruses have evolved a unique mechanism for nuclear egress of nascent progeny nucleocapsids: the nucleocapsids bud through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes (primary envelopment), and enveloped nucleocapsids then fuse with the outer nuclear membrane to release nucleocapsids into the cytoplasm (de-envelopment). We have shown that the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (or VP13/VP14) is a novel regulator for HSV-1 nuclear egress. In particular, we demonstrated the following: (i) UL47 formed a complex(es) with HSV-1 proteins UL34, UL31, and/or Us3, which have all been reported to be critical for viral nuclear egress, and these viral proteins colocalized at the nuclear membrane in HSV-1-infected cells; (ii) the UL47-null mutation considerably reduced primary enveloped virions in the perinuclear space although capsids accumulated in the nucleus; and (iii) UL47 was detected in primary enveloped virions in the perinuclear space by immunoelectron microscopy. These results suggested that UL47 promoted HSV-1 primary envelopment, probably by interacting with the critical HSV-1 regulators for viral nuclear egress and by modulating their functions. IMPORTANCELike other herpesviruses, herpes simplex virus 1 (HSV-1) has evolved a vesicle-mediated nucleocytoplasmic transport mechanism for nuclear egress of nascent progeny nucleocapsids. Although previous reports identified and characterized several HSV-1 and cellular proteins involved in viral nuclear egress, complete details of HSV-1 nuclear egress remain to be elucidated. In this study, we have presented data suggesting (i) that the major HSV-1 virion structural protein UL47 (or VP13/VP14) formed a complex with known viral regulatory proteins critical for viral nuclear egress and (ii) that UL47 played a regulatory role in HSV-1 primary envelopment. Thus, we identified UL47 as a novel regulator for HSV-1 nuclear egress.M orphogenesis of herpes simplex virus 1 (HSV-1), like that of other herpesviruses, takes place in two different cellular compartments (1, 2). Viral DNA replication and transcription, capsid assembly, and packaging of nascent progeny virus genomes into preformed capsids take place in the nucleus, and final envelopment takes place in the cytoplasm (1, 2). Since herpesvirus nucleocapsids are too large to traverse the nuclear lamina or cross the inner and outer nuclear membranes (INM and ONM, respectively) through nuclear pores, these viruses appear to have evolved a unique nuclear egress mechanism in which progeny nucleocapsids acquire primary envelopes by budding through the INM into the space between the INM and ONM, the perinuclear space, and then the enveloped nucleocapsids fuse with the ONM to release de-enveloped nucleocapsids into the cytoplasm (1, 2).In the present study, we focus on the first step of HSV-1 nuclear egress, the process by which progeny nucleocapsids acquire primary envelopes by budding through the INM into the perinuclear space (...

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 ...

The influenza A virus genome is composed of eight single-stranded negative-sense RNAs. Eight distinct viral RNA segments (vRNAs) are selectively packaged into progeny virions, with eight vRNAs in ribonucleoprotein complexes (RNPs) arranged in a specific “1+7” pattern, that is, one central RNP surrounded by seven RNPs. Here we report the genome packaging of an artificially generated seven-segment virus that lacks the hemagglutinin (HA) vRNA. Electron microscopy shows that, even in the presence of only seven vRNAs, the virions efficiently package eight RNPs arranged in the same “1+7” pattern as wild-type virions. Next-generation sequencing reveals that the virions specifically incorporate host-derived 18S and 28S ribosomal RNAs (rRNAs) seemingly as the eighth RNP in place of the HA vRNA. These findings highlight the importance of the assembly of eight RNPs into a specific “1+7” configuration for genome packaging in progeny virions and suggest a potential role for cellular RNAs in viral genome packaging.

Herpesviruses have evolved a unique mechanism for nucleocytoplasmic transport of nascent nucleocapsids: the nucleocapsids bud through the inner nuclear membrane (INM; primary envelopment), and the enveloped nucleocapsids then fuse with the outer nuclear membrane (de-envelopment). Little is known about the molecular mechanism of herpesviral de-envelopment. We show here that the knockdown of both CD98 heavy chain (CD98hc) and its binding partner ␤1 integrin induced membranous structures containing enveloped herpes simplex virus 1 (HSV-1) virions that are invaginations of the INM into the nucleoplasm and induced aberrant accumulation of enveloped virions in the perinuclear space and in the invagination structures. These effects were similar to those of the previously reported mutation(s) in HSV-1 proteins gB, gH, UL31, and/or Us3, which were shown here to form a complex(es) with CD98hc in HSV-1-infected cells. These results suggested that cellular proteins CD98hc and ␤1 integrin synergistically or independently regulated HSV-1 de-envelopment, probably by interacting directly and/or indirectly with these HSV-1 proteins. IMPORTANCECertain cellular and viral macromolecular complexes, such as Drosophila large ribonucleoprotein complexes and herpesvirus nucleocapsids, utilize a unique vesicle-mediated nucleocytoplasmic transport: the complexes acquire primary envelopes by budding through the inner nuclear membrane into the space between the inner and outer nuclear membranes (primary envelopment), and the enveloped complexes then fuse with the outer nuclear membrane to release de-enveloped complexes into the cytoplasm (de-envelopment). However, there is a lack of information on the molecular mechanism of de-envelopment fusion. We report here that HSV-1 recruited cellular fusion regulatory proteins CD98hc and ␤1 integrin to the nuclear membrane for viral de-envelopment fusion. This is the first report of cellular proteins required for efficient de-envelopment of macromolecular complexes during their nuclear egress. Herpesviruses are enveloped double-stranded DNA viruses that replicate their genomes and package the nascent progeny viral genomes into capsids in the nucleus, but these nascent viruses acquire their final envelopes in the cytoplasm (1, 2). Therefore, herpesvirus nucleocapsids must traverse the inner nuclear membrane (INM) and outer nuclear membrane (ONM) for viral morphogenesis. Since herpesvirus nucleocapsids are too large to cross the INM and ONM through nuclear pores, the viruses evolved a unique nuclear egress mechanism: progeny nucleocapsids acquire primary envelopes by budding through the INM into the perinuclear space between the INM and ONM (primary envelopment) and enveloped nucleocapsids then fuse with the ONM to release de-enveloped nucleocapsids into the cytoplasm (de-envelopment) (1, 2). Although this type of vesicle-mediated nucleocytoplasmic transport has not been reported previously, other than for herpesvirus nuclear egress, it has recently been reported that Drosophila cellular ribonu...