Involvement of the UL24 protein in herpes simplex virus 1-induced dispersal of B23 and in nuclear egress

UL24 of herpes simplex virus 1 (HSV-1) is widely conserved within the Herpesviridae family. Herein, we tested the hypothesis that UL24, which we have previously shown to induce the redistribution of nucleolin, also affects the localization of the nucleolar protein B23. We found that HSV-1-induced dispersal of B23 was dependent on UL24. The conserved N-terminal portion of UL24 was sufficient to induce the redistribution of B23 in transient transfection assays. Mutational analysis revealed that the endonuclease motif of UL24 was important for B23 dispersal in both transfected and infected cells. Nucleolar protein relocalization during HSV-1 infection was also observed in non-immortalized cells. Analysis of infected cells by electron microscopy revealed a decrease in the ratio of cytoplasmic versus nuclear viral particles in cells infected with a UL24-deficient strain compared to KOS-infected cells. Our results suggest that UL24 promotes nuclear egress of nucleocapsids during HSV-1 infection, possibly though effects on nucleoli.     

Characterization of molecular determinants for nucleocytoplasmic shuttling of PRV UL54

The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein and essential for HSV-1 infection. To determine if UL54 might shuttle between the nucleus and cytoplasm, as has been shown for its homologues in human herpesviruses, the molecular determinants for its nucleocytoplasmic shuttling were investigated. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of chromosome region maintenance 1 (CRM1). However, TAP/NXF1 promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP/NXF1, but not CRM1, dependent nuclear export pathway. Furthermore, UL54 was demonstrated to target to the nucleus through a classic Ran-, importin β1- and α5-dependent nuclear import mechanism.     

The role of protein kinase activity expressed by the UL13 gene of herpes simplex virus 1: the activity is not essential for optimal expression of UL41 and ICP0

Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that is packaged into virions and regulates optimal expression of ICP0 and a subset of late (gamma) proteins, including UL41 in infected cells. In the present study, we investigated the role(s) of the protein kinase activity of UL13 in viral replication using a recombinant virus expressing enzymatically inactive UL13 after an amino acid substitution in the invariant lysine of UL13. The recombinant virus carrying this mutation formed smaller plaques yielded 10-fold less progeny than wild-type virus but could not be differentiated from wild-type virus with respect to accumulation of UL41 and ICP0 in infected cells. These results indicate that the protein kinase activity of UL13 plays a role in viral replication in cell culture, but the activity is not essential for the optimal expression of UL41 and ICP0.     

Roles for herpes simplex virus type 1 UL34 and US3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress

Cells infected with wild type HSV-1 showed significant lamin A/C and lamin B rearrangement, while UL34-null virus-infected cells exhibited few changes in lamin localization, indicating that UL34 is necessary for lamin disruption. During HSV infection, US3 limited the development of disruptions in the lamina, since cells infected with a US3-null virus developed large perforations in the lamin layer. US3 regulation of lamin disruption does not correlate with the induction of apoptosis. Expression of either UL34 or US3 proteins alone disrupted lamin A/C and lamin B localization. Expression of UL34 and US3 together had little effect on lamin A/C localization, suggesting a regulatory interaction between the two proteins. The data presented in this paper argue for crucial roles for both UL34 and US3 in regulating the state of the nuclear lamina during viral infection.     

Herpes simplex virus 2 UL13 protein kinase disrupts nuclear lamins

Herpesviruses must cross the inner nuclear membrane and underlying lamina to exit the nucleus. HSV-1 US3 and PKC can phosphorylate lamins and induce their dispersion but do not elicit all of the phosphorylated lamin species produced during infection. UL13 is a serine threonine protein kinase conserved among many herpesviruses. HSV-1 UL13 phosphorylates US3 and thereby controls UL31 and UL34 nuclear rim localization, indicating a role in nuclear egress. Here, we report that HSV-2 UL13 alone induced conformational changes in lamins A and C and redistributed lamin B1 from the nuclear rim to intranuclear granular structures. HSV-2 UL13 directly phosphorylated lamins A, C, and B1 in vitro, and the lamin A1 tail domain. HSV-2 infection recapitulated the lamin alterations seen upon expression of UL13 alone, and other alterations were also observed, indicating that additional viral and/or cellular proteins cooperate with UL13 to alter lamins during HSV-2 infection to allow nuclear egress.     

Substrate specificity of the herpes simplex virus type 2 UL13 protein kinase

The UL13 protein kinase is conserved among many herpesviruses but HSV-2 UL13 specificity is not known. Here, we found that HSV-2 UL13 is a phosphoprotein that autophosphorylates, and that serines within ERK and Cdc2 motifs were important for autophosphorylation but not for UL13 phosphorylation of exogenous substrates. HSV-2 UL13 phosphorylated a peptide also recognized by ERK and Cdc2. However, mutation of substrate residues critical for Cdc2 or Erk phosphorylation did not alter HSV-2 UL13 phosphorylation of the peptide, and HSV-2 UL13 did not phosphorylate standard Cdc2 or Erk peptide substrates. Mutation of prolines surrounding the peptide phosphoacceptor site reduced phosphorylation by HSV-2 UL13, and a peptide containing serine-proline amid alanines and glycines was phosphorylated. Thus, HSV-2 UL13 does not mimic ERK or Cdc2 substrate recognition and its minimal recognition motif can be serine-proline. This motif's simplicity indicates that distal sequence or protein structure contributes to HSV-2 UL13 substrate specificity.     

Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina

The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress.     

Genomic variation among herpes simplex virus type 1 strains: virus DNA analysis of isolates from Saudi patients.

Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes HindIII and BamHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with HindIII restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BamHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise to the observed DNA polymorphism were mapped to the unique region of the long and short segments of the genome.     

Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

Previous results indicated that the herpes simplex virus 1 (HSV-1) U(L)31 gene is necessary and sufficient for localization of the U(L)34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U(L)31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U(L)31 gene. The replication of the U(L)31 deletion virus was restored on U(L)31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U(L)34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U(L)34 protein localized at the nuclear membrane in rabbit skin cells, and U(L)31 complementing CV1 cells infected with the U(L)31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U(L)34 protein to the nuclear membrane. We speculate that this function partially complements that of U(L)31 and may explain why U(L)31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.     

Site-directed mutagenesis in the active site of the herpes simplex virus type 1 thymidine kinase gene

The thymidine kinase (TK) of herpes simplex virus type 1 (HSV-1) contains three regions of homology to other ATP utilizing enzymes. We have altered one region of the protein, which seems to play an important role in phosphorylating substrates by site-directed mutagenesis. When the aspartate 162 was changed to asparagine, the enzyme lost its activity. To identify the inactive protein, expressed by a vaccinia vector in eukaryotic cells, a monospecific antiserum against a bacterial tryptophan E-HSV-1 TK fusion protein was made. These results support the suggestion that aspartate 162 is essential for the enzymatic activity.     

The immune response to herpes simplex virus: comparison of the specificity and relative titers of serum antibodies directed against viral polypeptides following primary herpes simplex virus type 1 infections

Employing an immunoblotting technique, the polypeptide specificity and relative titers of anti-HSV IgG reactive with denaturation-resistant epitopes on HSV proteins were determined in patients experiencing primary HSV-1 infections at various anatomical sites. Early sera from previously seronegative patients with primary HSV-1 infections were found to have comparatively low levels of antibody directed against the major viral glycoprotein antigens (gB, gC, and gD) relative to titers present in sera of individuals with long-standing, latent orofacial HSV-1 infections. Patients with primary infections did however have high titers of antibody directed against a series of low molecular weight HSV polypeptide antigens. These antigens were found to be antigenically related to a structural component of virion nucleocapsids. At later times postinfection, titers of antibodies directed against other viral polypeptides including the major glycoproteins increased to levels more closely approximating those observed in latently infected individuals. These results indicate that the anti-HSV IgG detected by immunoblot analysis which appears earliest following primary infection is not directed against the known major infected cell or virion glycoprotein surface antigens but rather against an internal capsid protein of HSV.     

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

A role for heparan sulfate 3-O-sulfotransferase isoform 2 in herpes simplex virus type 1 entry and spread

Heparan sulfate (HS) 3-O-sulfotransferase isoform-2 (3-OST-2), which belongs to a family of enzymes capable of generating herpes simplex virus type-1 (HSV-1) entry and spread receptors, is predominantly expressed in human brain. Despite its unique expression pattern, the ability of 3-OST-2 to mediate HSV-1 entry and cell-to-cell fusion is not known. Our results demonstrate that expression of 3-OST-2 can render Chinese hamster ovary K1 (CHO-K1) cells susceptible to entry of wild-type and mutant strains of HSV-1. Evidence for generation of gD receptors by 3-OST-2 were suggested by gD-mediated interference assay and the ability of 3-OST-2-expressing CHO-K1 cells to preferentially bind HSV-1 gD, which could be reversed by prior treatment of cells with HS lyases (heparinases II/III). In addition, 3-OST-2-expressing CHO-K1 cells acquired the ability to fuse with cells-expressing HSV-1 glycoproteins, a phenomenon that mimics a way of viral spread in vivo. Demonstrating specificity, the cell fusion was inhibited by soluble 3-O-sulfated forms of HS, but not unmodified HS. Taken together, our results raise the possibility of a role of 3-OST-2 in the spread of HSV-1 infection in the brain.     

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.     

Complex mechanisms for the packaging of the UL16 tegument protein into herpes simplex virus

The conserved UL16 tegument protein of herpes simplex virus exhibits dynamic capsid-binding properties with a release mechanism that is triggered during initial virus attachment events. In an effort to understand the capsid association and subsequent release of UL16, we sought to define the mechanism by which this protein is packaged into virions. The data presented here support a model for the addition of some UL16 to capsids prior to their arrival at the TGN. UL16 was found on capsids isolated from cells infected with viruses lacking UL36, UL37 or gE/gD, which are defective for budding and accumulate non-enveloped capsids in the cytoplasm. Additionally, membrane-flotation experiments showed that UL16 co-purified with cytoplasmic capsids that are not associated with membranes. Moreover, the amount of UL16 packaged into extracellular particles was severely reduced in the absence of two conserved binding partners, UL21 or UL11.     

Analysis of the herpes simplex virus type 1 UL6 gene in patients with stromal keratitis

Recent work suggests that herpes simplex virus (HSV) stromal keratitis in the mouse is caused by autoreactive T lymphocytes triggered by a 16 amino acid region of the HSV UL6 protein (aa299-314), Science 279, 1344-1347). In the present study we sought to determine whether genetic variation of this presumed autoreactive UL6 epitope is responsible for different pathogenic patterns of human HSV keratitis. To accomplish this, we sequenced the HSV UL6 gene from ocular isolates of 10 patients with necrotizing stromal keratitis, 7 patients with recurrent epithelial keratitis, and 8 patients with other forms of HSV keratitis. The sequences obtained predicted identical UL6(299-314) epitopes for all 25 viral isolates. Furthermore, the upstream sequence of all isolates was free of insertions, deletions, and stop codons. We conclude that different pathogenic patterns of human HSV keratitis occur independent of genetic variation of the HSV UL6 (299-314) epitope.     

Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling

Host cells respond to viral infection by many mechanisms, including the production of type I interferons which act in a paracrine and autocrine manner to induce the expression of antiviral interferon-stimulated genes (ISGs). Viruses have evolved means to inhibit interferon signaling to avoid induction of the innate immune response. Herpes simplex virus 1 (HSV-1) has several mechanisms to inhibit type I interferon production, the activities of ISGs, and the interferon signaling pathway itself. We report that the inhibition of the Jak/STAT pathway by HSV-1 requires viral gene expression and that viral immediate-early protein ICP27 plays a role in downregulating STAT-1 phosphorylation and in preventing the accumulation of STAT-1 in the nucleus. We also show that expression of ICP27 by transfection causes an inhibition of IFN-induced STAT-1 nuclear accumulation. Therefore, ICP27 is necessary and sufficient for at least some of the effects of HSV infection on STAT-1.     

Reconstitution of herpes simplex virus type 1 ribonucleotide reductase activity from the large and small subunits

An assay for the presence of functional large (RR₁) and small (RR₂) subunits of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been developed. The system utilizes two temperature-sensitive mutants, ts1207, which has a lesion in RR₁, and ts1222, which has a lesion in RR₂. In cells infected with ts1207 at 39.5°C, the defective RR₁ is unable to associate with RR₂ to form an active enzyme, and, as a result, a pool of functional RR₂ and defective RR₁ accumulates. Evidence presented in this paper suggest that cells infected with ts1222 at either 31°C or 39.5°C accumulate a pool of functional RR₁, but do not contain detectable RR₂. Virus-specific ribonucleotide reductase activity was produced in cells coinfected with both mutants at 39.5°C, each virus contributing one functional subunit to the holoenzyme. No enzyme activity was detected in cells infected with each mutant alone at this temperature. When partially purified extracts of cells infected with ts1207 at the nonpermissive temperature were mixed with those from ts1222-infected cells, a fully functional enzyme was also formed. These results demonstrate that HSV-1 ribonucleotide reductase activity can be reconstituted both in vivo and in vitro from the nondefective subunits produced by ts1222 and ts1207.     

ICP27-dependent resistance of herpes simplex virus type 1 to leptomycin B is associated with enhanced nuclear localization of ICP4 and ICP0

It was previously shown that herpes simplex virus type 1 (HSV-1) is sensitive to leptomycin B (LMB), an inhibitor of nuclear export factor CRM1, and that a single methionine to threonine change at residue 50 (M50T) of viral immediate-early (IE) protein ICP27 can confer LMB resistance. In this work, we show that deletion of residues 21-63 from ICP27 can also confer LMB resistance. We further show that neither the M50T mutation nor the presence of LMB affects the nuclear shuttling activity of ICP27, suggesting that another function of ICP27 determines LMB resistance. A possible clue to this function emerged when it was discovered that LMB treatment of HSV-1-infected cells dramatically enhances the cytoplasmic accumulation of two other IE proteins, ICP0 and ICP4. This effect is completely dependent on ICP27 and is reversed in cells infected with LMB-resistant mutants. Moreover, LMB-resistant mutations in ICP27 enhance the nuclear localization of ICP0 and ICP4 even in the absence of LMB, and this effect can be discerned in transfected cells. Thus, the same amino (N)-terminal region of ICP27 that determines sensitivity to LMB also enhances ICP27's previously documented ability to promote the cytoplasmic accumulation of ICP4 and ICP0. We speculate that ICP27's effects on ICP4 and ICP0 may contribute to HSV-1 LMB sensitivity.     

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.