Herpes simplex virus type 1 entry is inhibited by the cobalt chelate complex CTC-96.

The CTC series of cobalt chelates display in vitro and in vivo activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). The experiments described here identify the stage in the virus life cycle where CTC-96 acts and demonstrate that the drug inhibits infection of susceptible cells. CTC-96 at 50 microg/ml has no effect on adsorption of virions to Vero cell monolayers. Penetration assays reveal that CTC-96 inhibits entry of the virus independent of gC and cellular entry receptors. This observation was supported by the failure to detect the accumulation of virus-specified proteins and alpha mRNA transcripts when CTC-96 is present at the onset of infection. Moreover, virion-associated alphaTIF does not accumulate in the nucleus of cells infected in the presence of CTC-96. CTC-96 targets the initial fusion event between the virus and the cell and also inhibits cell-to-cell spread and syncytium formation. Furthermore, CTC-96 inhibits plaque formation by varicella-zoster virus and vesicular stomatitis virus as efficiently as by HSV-1. Collectively, these experiments suggest that CTC-96 is a broad-spectrum inhibitor of infection by enveloped viruses and that it inhibits HSV-1 infection at the point of membrane fusion independent of the type of virus and cellular receptors present.

Herpes simplex virus IE63 (ICP27) protein interacts with spliceosome-associated protein 145 and inhibits splicing prior to the first catalytic step.

The multifunctional herpes simplex virus type 1 (HSV-1) protein IE63 (ICP27) interacts with the essential pre-mRNA splicing factor, spliceosome-associated protein 145 (SAP145), and in infected cells IE63 and SAP145 colocalize. This interaction was reduced or abrogated completely using extracts from cells infected with IE63 viral mutants, with mutations in IE63 KH and Sm homology domains, which do not exhibit host shutoff or inhibit splicing. In the presence of IE63, splicing in vitro was inhibited prior to the first catalytic step and the B/C complex formed during splicing was shifted up in mobility and reduced in intensity. With the use of splicing extracts, IE63 and SAP145 both comigrated with the B/C complex, suggesting that they interact within this complex to inhibit B/C complex formation or conversion. The inhibition of splicing may facilitate the export of viral or cellular transcripts, possibly via other protein partners of IE63. These data provide important new insights into how IE63 influences pre-mRNA processing during HSV-1 infection.

Orientation of a novel DNA binding site affects human papillomavirus-mediated transcription and replication.

A consensus binding site for the human papillomavirus (HPV) E2 protein was determined from an unbiased set of degenerate oligonucleotides using cyclic amplification and selection of targets (CASTing). Detectable DNA-protein complexes were formed after six to nine cycles of CASTing. A population of selected binding sites was cloned, and a consensus was determined by statistical analysis of the DNA sequences of individual isolates. Starting from a pool with 20 random bases, a consensus binding site of ACAC-N(5)-GGT was derived. CASTing and electrophoretic mobility shift analyses demonstrate that human but not bovine papillomavirus E2 proteins recognize this sequence. The presence of this sequence in papillomavirus genomes suggests a role for its function. We demonstrate that this site functionally substitutes for the canonical E2 binding site (ACCG-N(4)-CGGT) in both transient-transcription and DNA replication assays. This sequence, in most instances, is interchangeable with the resident E2 binding sites in the context of the HPV type 16 long control region. Where the novel sequence does not support E2-mediated effects on gene expression or DNA replication, we demonstrate that changing the orientation of the novel sequence restores this effect.

Gps2, a protein partner for human papillomavirus E6 proteins.

We have used the yeast two-hybrid system to screen a cDNA library prepared from normal human epidermal keratinocytes and identified protein partners for human papilloma virus (HPV) E6 proteins. A clone that encoded Gps2 interacted with E6 proteins from HPVs of high and low oncogenic risk. The specificity of these reactions was verified and the regions of E6 that were required for interaction were mapped. Steady-state and pulse-chase analyses of cells cotransfected with DNAs expressing E6 from either HPV6 or HPV18 and Gps2 demonstrated that the E6 proteins induced the degradation of Gps2 in vivo but not in vitro. Gps2 exhibited transcriptional activation activity, and high-risk E6 suppressed this activity.

Open reading frame S/L of varicella-zoster virus encodes a cytoplasmic protein expressed in infected cells.

We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3' untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.

Identification of an export control sequence and a requirement for the KH domains in ICP27 from herpes simplex virus type 1.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an RNA-binding protein that performs multiple functions required for the expression of HSV-1 genes during a productive infection. One essential function involves shuttling between the nucleus and the cytoplasm. Some of the domains identified in ICP27 include a leucine-rich nuclear export sequence (NES), a nuclear localization signal, three KH-like RNA-binding domains, and an RGG-box type RNA-binding motif. To study the contribution of two of the essential domains in ICP27 to HSV gene expression, we generated recombinant herpesviruses carrying deleterious mutations in the NES and KH domains of ICP27. To accomplish this, we fused the green fluorescent protein (GFP) to ICP27 and utilized fluorescence as a marker to isolate recombinant herpesviruses. Fusion of GFP to wild-type ICP27 did not disturb its localization or function or significantly reduce virus yield. Analysis of HSV gene expression in cells infected with a recombinant virus carrying a point mutation in the first KH-like RNA-binding domain revealed that nuclear export of ICP27 was not blocked, and the expression of only a subset of ICP27-dependent late genes was affected. These findings suggest that individual KH-like RNA-binding motifs in ICP27 may be involved in binding distinct RNAs. Analysis of recombinant viruses carrying a lethal mutation in the NES of ICP27 was not accomplished because this mutation results in a strong dominant-negative phenotype. Finally, we demonstrate that shuttling by ICP27 is regulated by an export control sequence adjacent to its NES that functions like the inhibitory sequence element found adjacent to the NES of NS1 from influenza virus.

Herpesvirus mRNAs are sorted for export via Crm1-dependent and -independent pathways.

Cellular pre-mRNA splicing is inhibited by ICP27, a herpes simplex virus regulatory protein, resulting in the shutoff of host protein synthesis. Here we reveal that ICP27 also mediates the export of some virus RNAs via a Crm1-dependent pathway and present evidence that independent domains are required for these functions. Sorting of some viral mRNAs for nuclear export requires Crm1, while other virus mRNAs are exported via another pathway.

Varicella-zoster virus proteins in skin lesions: implications for a novel role of ORF29p in chickenpox.

Skin biopsy samples from varicella-zoster virus (VZV)-infected patients examined by immunohistochemistry demonstrated VZV replication in nonepithelial cell types. ORF29p, a nonstructural nuclear protein, was found in nerves of two of six patients with chickenpox. In tissue culture, ORF29p was secreted by VZV-infected fibroblasts. Extracellular ORF29p can be taken up through endocytosis by human neurons, implying a novel role for this protein in pathogenesis.

Enhanced production of mineralized nodules and collagenous proteins in vitro by calcium ascorbate supplemented with vitamin C metabolites.

BACKGROUND: Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate). METHODS: Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence. RESULTS: In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate. CONCLUSIONS: In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.

The host-cell architectural protein HMG I(Y) modulates binding of herpes simplex virus type 1 ICP4 to its cognate promoter.

The productive infection cycle of herpes simplex virus is controlled in part by the action of ICP4, an immediate-early gene product that acts as both an activator and repressor of transcription. ICP4 is autoregulatory, and IE-3, the gene that encodes it, contains a high-affinity binding site for the protein at its cap site. Previously, we had demonstrated that this site could be occupied by proteins found in nuclear extracts from uninfected cells. A HeLa cell cDNA expression library was screened with a DNA probe containing the IE-3 gene cap site, and clones expressing the architectural chromatin proteins HMG I and HMG Y were identified by this technique. HMG I is shown to augment binding of ICP4 to its cognate site in in vitro assays and to enhance the activity of this protein in short-term transient expression assays.

The NH2 terminus of the herpes simplex virus type 1 regulatory protein ICP0 contains a promoter-specific transcription activation domain.

The transcriptional program of herpes simplex virus is regulated by the concerted action of three immediate-early (alpha) proteins, ICP4, ICP27, and ICP0. The experiments described in this study examine the role of the acidic amino terminus (amino acids 1 to 103) of ICP0 in gene activation. When tethered to a DNA binding domain, this sequence activates transcription in the yeast Saccharomyces cerevisiae. Deletion of these amino acids affects the ability of ICP0 to activate alpha-gene promoter reporters in transient expression assays, while it has little or no effect on a beta- and a gamma-gene reporter in the same assay. Viruses that express the deleted form of ICP0 (ICP0-NX) have a small-plaque phenotype on both Vero cells and the complementing cell line L7. Transient expression and immunofluorescence analyses demonstrate that ICP0-NX is a dominant negative form of ICP0. Immunoprecipitation of ICP0 from cells coinfected with viruses expressing ICP0-NX and ICP0 revealed that ICP0 oligomerizes in infected cells. These data, in conjunction with the finding that ICP0-N/X is dominant negative, provide both biochemical and genetic evidence that ICP0 functions as a multimer in infected cells.

Aberrant intracellular localization of Varicella-Zoster virus regulatory proteins during latency.

Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus.

Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is required posttranscriptionally for the expression of HSV-1 late genes during a productive infection. ICP27 also inhibits host cell pre-mRNA splicing, effectively shutting off host cell protein synthesis. Here we describe intragenic suppressors of LG4, a virus with a conditional lethal mutation in the gene encoding ICP27. At the restrictive temperature, tsICP27 from LG4 fails to inhibit host cell pre-mRNA splicing and to activate the expression of HSV-1 late-gene products. Although the suppressors of LG4 restore virus growth, they still fail to inhibit host cell pre-mRNA splicing. Thus, the role of ICP27 in the synthesis of late proteins is independent of host shutoff. In HSV-1-infected cells, ICP27 shuttles between the nucleus and the cytoplasm. Shuttling of ICP27 occurs only at late times during infection. In transfected cells, ICP27 shuttling was dependent on coexpression of RNA from a late HSV-1 gene. While shuttling does not occur in cells infected with LG4 at 39.5 degrees C, the suppressors of LG4 restore shuttling. Temperature shift experiments correlate the defect in shuttling with the temperature-sensitive phenotype of LG4. These data provide a correlation between shuttling of ICP27 and the expression of HSV-1 late-gene products. We propose that ICP27 regulates late-gene protein synthesis by facilitating the export of late RNAs.

Physical and functional interactions between herpes simplex virus immediate-early proteins ICP4 and ICP27.

The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediate-early proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the alpha4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4.

In situ hybridization detection of varicella zoster virus in paraffin-embedded skin biopsy samples.

BACKGROUND: When virologic and molecular diagnostic techniques are unavailable, the diagnosis of varicella zoster virus (VZV) infection depends on clinical criteria and histologic evaluation of skin biopsy specimens or Tzank preparations. These methods can misdiagnose chickenpox and zoster, particularly when the clinical manifestations are atypical. OBJECTIVE: To improve diagnosis in these settings, we developed an in situ hybridization technique for the detection of VZV utilizing a fluorescein-labeled oligonucleotide probe visualized with anti-fluorescein alkaline phosphatase-conjugated antibody. STUDY DESIGN: We retrospectively examined 26 paraffin-embedded skin biopsy specimens with histologic features consistent with VZV or herpes simplex virus (HSV) infection and 11 control cases by in situ hybridization. In situ hybridization for VZV and HSV-1 was compared with polymerase chain reaction (PCR) for VZV and HSV-1 and clinical and histologic examination. RESULTS: Thirteen of the 26 study cases and two of the 11 control cases were positive for VZV by in situ hybridization. When compared with PCR, in situ hybridization was 92% sensitive and 88% specific. When compared with clinical diagnosis, in situ hybridization was 86% sensitive and 87% specific. All cases of chickenpox had VZV-positive inflammatory cells in the dermis but this finding was less frequent among the cases of zoster. CONCLUSIONS: This in situ hybridization technique is a sensitive and specific method for the diagnosis of VZV in skin lesions that is applicable to most histopathology laboratory settings. In addition, in situ hybridization reveals individual infected cells and may provide insight into the pathogenesis of VZV skin infection.

Reactivated and latent varicella-zoster virus in human dorsal root ganglia.

Ganglia obtained at autopsy were examined by in situ hybridization from one patient with zoster (also called herpes zoster or shingles), two varicella-zoster virus (VZV)-seropositive patients with clinical evidence of zoster, one VZV-seronegative child, and one fetus. Ganglia positive for VZV had a hybridization signal in both neuronal and nonneuronal satellite cells. Ganglia obtained from the fetus and from the seronegative infant were consistently negative for VZV. Two striking observations were evident regarding the presence of VZV DNA in ganglia obtained from the individual with zoster at the time of death. First, ganglia innervating the sites of reactivation and ganglia innervating adjacent sites yielded strongly positive signals in neurons and satellite cells, whereas ganglia from distant sites were rarely positive. Second, VZV DNA was found in both the nuclei and the cytoplasm of neurons innervating areas of zoster. However, in neurons innervating zoster-free areas, VZV DNA was found only in the nucleus of neurons and their supporting satellite cells. Immunohistochemistry with a fluorescent monoclonal antibody to the VZV glycoprotein gpI, a late virus protein, revealed a positive signal in the cytoplasm of ganglia with clinical evidence of reactivation. These results illustrate that both neuronal and satellite cells become latently infected following primary VZV infection. The presence of VZV DNA and gpI in the cytoplasm of neurons demonstrates productive infection following reactivation at the site of latency.

pALEX, a dual-tag prokaryotic expression vector for the purification of full-length proteins.

pALEX, a prokaryotic expression vector, was constructed in which the multiple cloning site (MCS, polylinker) is flanked by sequences encoding glutathione S-transferase (GST) at the 5' end and a His6 residue tag at the 3' end. Open reading frames cloned into this vector can direct production of fusion proteins with GST at their N terminus and a His6 tag at their C terminus. This allows for the purification of full-size fusion proteins by a sequential two-step procedure on glutathione-agarose and Ni(2+)-agarose columns.

Polyamines alter sequence-specific DNA-protein interactions.

The polyamines are abundant biogenic cations implicated in many biological processes. Despite a plethora of evidence on polyamine-induced DNA conformational changes, no thorough study of their effects on the activities of sequence-specific DNA binding proteins has been performed. We describe the in vitro effects of polyamines on the activities of purified, representative DNA-binding proteins, and on complex protein mixtures. Polyamines at physiological concentrations enhance the binding of several proteins to DNA (e.g. USF, TFE3, Ig/EBP, NF-IL6, YY1 and ICP-4, a herpes simplex virus gene regulator), but inhibit others (e.g. Oct-1). The degree of enhancement correlates with cationic charge; divalent putrescine is ineffective whereas tetravalent spermine is more potent than trivalent spermidine. Polyamine effects on USF and ICP-4 result from increased rate of complex formation rather than a decreased rate of dissociation. DNAse I footprint analysis indicated that polyamines do not alter DNA-protein contacts. Polyamines also facilitate formation of complexes involving binding of more than one protein on a DNA fragment.

Isolation and characterization of an Escherichia coli DnaK mutant with impaired ATPase activity.

A temperature-sensitive mutant of DnaK, the principal Escherichia coli member of the 70 kDa heat shock protein family, has been isolated. The mutation, dnaK25, lies in the putative ATP binding pocket of DnaK. It consists of a C to T transition that changes the highly conserved proline 143 to serine. Mutant strains do not support the propagation of bacteriophage lambda or of plasmids that require DnaA for replication. They are also defective in the utilization of mannose and sorbitol. ATPase activity of the mutant protein is reduced 20-fold relative to wild-type, while autophosphorylation is unaffected. DnaK25 has a fourfold faster rate of nucleotide exchange than wild-type DnaK; nucleotide exchange by both proteins is markedly increased by GrpE. The DnaK25 ATPase is still stimulated by DnaJ and GrpE and by peptide substrates. However, the affinity of most peptides tested for stimulating the DnaK25 ATPase is reduced significantly. These results indicate that a mutation in the N-terminal nucleotide binding domain can alter substrate interactions with the C-terminal substrate binding site. Nucleotide exchange by both wild-type DnaK and DnaK25 proceeds at a much faster rate than ATP hydrolysis, and therefore cannot be the rate limiting step of ATP hydrolysis under the conditions used in these experiments. Consistent with this, peptides, which stimulate ATP hydrolysis, have no effect on nucleotide exchange. Peptides thus appear to stimulate the ATPase by acting at another step, such as increasing the rate of phosphate bond cleavage.

Inhibition of DnaK autophosphorylation by heat shock proteins and polypeptide substrates.

DnaK, the Hsp70 of Escherichia coli, autophosphorylates in vitro. Of the two heat shock proteins that interact with DnaK, GrpE inhibits DnaK phosphorylation, whereas DnaJ has no effect on the reaction. Three synthetic peptides are shown to inhibit DnaK phosphorylation. The potency of a given peptide correlates with its affinity for the DnaK protein. A truncated DnaK that lacks the carboxyl-terminal peptide-binding domain autophosphorylates; this reaction is resistant to the inhibitory peptides. Phosphorylation of the truncated DnaK is still inhibited by GrpE, indicating that the GrpE-binding site resides in the DnaK amino-terminal domain. Thus, DnaK phosphorylation is regulated in vitro, and possibly in vivo, by physiologically relevant substrates and cofactors.