The ICP34.5 protein of herpes simplex virus type 1 is a neurovirulence factor that plays critical roles in viral replication and anti-host responses. One of its functions is to recruit protein phosphatase 1 (PP1) that leads to the dephosphorylation of the ␣ subunit of translation initiation factor eIF2 (eIF2␣), which is inactivated by infection-induced phosphorylation. As PP1 is a protein phosphatase with a wide range of substrates, the question remains to be answered how ICP34.5 directs PP1 to specifically dephosphorylate eIF2␣. Here we report that ICP34.5 not only binds PP1 but also associates with eIF2␣ by in vitro and in vivo assays. The binding site of eIF2␣ is identified at amino acids 233-248 of ICP34.5, which falls in the highly homologous region with human gene growth arrest and DNA damage 34. The interaction between ICP34.5 and eIF2␣ is independent of the phosphorylation status of eIF2␣ at serine 51. Deletion mutation of this region results in the failure of dephosphorylation of eIF2␣ by PP1 and, consequently, interrupts viral protein synthesis and replication. Our data illustrated that the binding between viral protein ICP34.5 and the host eIF2␣ is crucial for the specific dephosphorylation of eIF2␣ by PP1. We propose that herpes simplex virus protein ICP34.5 bridges PP1 and eIF2␣ via their binding motifs and thereby facilitates the protein synthesis and viral replication.Viral infection can activate a series of host immune responses. One of the essential responses is the interruption of the viral protein synthesis, which is executed by doublestranded RNA-dependent protein kinase (PKR) (1). PKR is induced and activated upon viral infection and leads to the phosphorylation of the ␣ subunit of translation initiation factor eIF2 (eIF2␣) 2 at serine 51 (2). The phosphorylation of eIF2␣ globally inhibits the synthesis of viral proteins and cellular proteins (3), thus halting the viral replication.Many viruses have evolved strategies to counteract the antiviral response of PKR (4). For example, PKR activity can be inhibited by HIV-encoded Tar (5), hepatitis C virus NS5A (6), influenza virus NS1 (7), and so forth. In addition to affecting PKR activation as mentioned above, HSV-1 adopted mechanisms not only inhibiting PKR by Us11 (8) but also reversing the biochemical reaction catalyzed by PKR with its neurovirulent factor ICP34.5 (9).ICP34.5 is encoded by the ␥ 1 34.5 gene of HSV-1 and HSV-2. The HSV-1(F) ICP34.5 consists of 263 amino acids and can be divided into three domains: an amino-terminal domain, a linker region of ATP (Ala-Thr-Pro) repeats, and a carboxyl-terminal domain (9, 10). The function of the amino-terminal domain is implicated in the control of TBK1-mediated signaling (11) and also related to autophagy (12). The linker region, with a varying number of the Ala-Thr-Pro repeats, may affect the protein localization (13). The carboxyl-terminal domain is a stretch of 84 amino acids containing a consensus binding motif (R/KVXF) for protein phosphatase 1 (PP1) followed by an Ala-Arg-rich motif and i...

Background: HSV disrupts nuclear lamina for release from nucleus during productive infection. Results: A cellular protein, p32, contributes to the release of HSV from nucleus. Conclusion: p32 is hijacked by viral protein ICP34.5 to facilitate HSV nuclear egress and growth. Significance: The discovery of a novel target for viral protein provides insight for viral propagation.

The ␥ 1 34.5 protein of herpes simplex virus 1 is an essential factor for viral virulence. In infected cells, this viral protein prevents the translation arrest mediated by double-stranded RNA-dependent protein kinase R. Additionally, it associates with and inhibits TANK-binding kinase 1, an essential component of Toll-like receptor-dependent and -independent pathways that activate interferon regulatory factor 3 and cytokine expression. Here, we show that ␥ 1 34.5 is required to block the maturation of conventional dendritic cells (DCs) that initiate adaptive immune responses. Unlike wild-type virus, the ␥ 1 34.5 null mutant stimulates the expression of CD86, major histocompatibility complex class II (MHC-II), and cytokines such as alpha/beta interferon in immature DCs. Viral replication in DCs inversely correlates with interferon production. These phenotypes are also mirrored in a mouse ocular infection model. Further, DCs infected with the ␥ 1 34.5 null mutant effectively activate naïve T cells whereas DCs infected with wild-type virus fail to do so. Type I interferon-neutralizing antibodies partially reverse virus-induced upregulation of CD86 and MHC-II, suggesting that ␥ 1 34.5 acts through interferon-dependent and -independent mechanisms. These data indicate that ␥ 1 34.5 is involved in the impairment of innate immunity by inhibiting both type I interferon production and DC maturation, leading to defective T-cell activation.

Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required for their function are unclear. Here we report that the ␥ 1 34.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32, and protein kinase C. Unlike wild-type virus, an HSV mutant devoid of ␥ 1 34.5 or its amino terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild-type virus recruits protein kinase C to the nuclear membrane and triggers its activation, whereas the ␥ 1 34.5 mutants fail to exert such an effect. Accordingly, the ␥ 1 34.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells ␥ 1 34.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino terminus from ␥ 1 34.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by ␥ 1 34.5 promotes HSV replication. IMPORTANCEHSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus-infected cells. We report that the ␥ 1 34.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C, and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection. Herpes simplex virus 1 (HSV-1) replicates and packages its DNA in the cell nucleus. Once assembled, the nucleocapsids traverse the nucleoplasm and cross the nuclear lamina. The capsids bud through the nuclear membranes in a two-step process called envelopment and de-envelopment (1). In this process, the nuclear egress complex, consisting of UL31 and UL34, mediates vesiculation of the inner nuclear membrane and results in enveloped virions in the perinuclear space. Primary virions fuse with the outer nuclear membrane, which releases the capsids to the cytoplasm for further maturation (2). Accumulating evidence suggests that additional proteins, including Us3, ICP22, UL47, gB, and gH, coordinate with the UL31/34 complex to facilitate nuclear egress in infected cells (3-6).The nuclear lamina is a dense meshwork underlying the inner nuclear membrane (7). It is composed primarily of type V intermediate filament proteins, lamin A/C and lamin B. Besides providing structural support to the nucleus, the nuclear lamina potentially presents a barrier to the transit of virus capsids. A number of studies suggest that herpesviruses alter the nuclear lamina to promote nuclear egress (8-11). For example, HSV-1 activates protein kinase C (PKC) isoforms and induces phosphorylation of lamin B, which is dependent on the UL31/UL34 complex (1...

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor ( Stimulation of the T cell antigen receptor (TCR)2 initiates a cascade of signaling events that lead to T cell activation. Calcium plays a central role in this process and has been studied intensively (1-4). Engagement of TCR triggers the activation and accumulation of enzymes and adapter molecules to the proximal membrane (5, 6), such as tyrosine phosphorylation and activation of phospholipase-C␥1 (PLC-␥1), thereby increasing the production of inositol 1,4,5-trisphosphate (IP 3 ). IP 3 binds to and activates the inositol 1,4,5-trisphosphate receptor (IP 3 R), which results in Ca 2ϩ release from the endoplasmic reticulum (ER) and the subsequent calcium influx from Ca 2ϩ release-activated Ca 2ϩ channel (CRAC) (1, 7). The elevated cytoplasmic [Ca 2ϩ ] i evokes a multitude of cellular responses, such as the NFAT-mediated gene expressions and the cell proliferation (8, 9).Vav1 is expressed specifically in hematopoietic cells as a 95-kDa protein, which plays pivotal roles as a guanine exchange factor (GEF) for small GTPases as well as a scaffold protein in the activation of hematopoietic cells (10 -12). The importance of Vav1 is because of its multiple structural elements, including a calponin homology (CH) domain, an acidic motif, a Dbl homology domain, a pleckstrin homology (PH) domain, a cysteine-rich motif, and one single SH2 domain flanked by two SH3 domains responsible for signaling protein assembly (12, 13). Upon TCR engagement, Vav1 is phosphorylated on the key tyrosine residues in the acidic motif, leading to the exposure of active Dbl homology domain for GDP/GTP exchange activity (14). Studies on vav1 Ϫ/Ϫ T cells isolated from knock-out mice demonstrated that Vav1 is essential for normal T cell activation and proliferation (15)(16)(17). In addition, the vav1-null cell line, J.Vav1, derived from Jurkat cells by somatic gene targeting approach, also exhibits pleiotropic defects in TCR-mediated signaling pathways (18).T cell stimulation evokes a biphasic calcium flux as follows: calcium release from intracellular stores followed by calcium influx across the plasma membrane (7,19). IP 3 Rs dominantly control the initiation of IP 3 -induced calcium release, demonstrated by using antisense knockdown of IP 3 R to block calcium release from the ER (20). Jurkat T cells express three IP 3 R isoforms, IP 3 R-1, IP 3 R-2, and IP 3 R-3 (21), which differ significantly in their sensitivity to IP 3 (22,23). A tyrosine kinase, Fyn, was suggested to modulate IP 3 R channel activities (24,25). Interactions between IP 3 R and other proteins, such as calmodulin (CaM), were reported to control the channel opening. Although some observations viewed CaM as an inhibitory protein of IP 3 R (26 -28), more...