Relief of pain induced by varicella-zoster virus in a rat model of post-herpetic neuralgia using a herpes simplex virus vector expressing enkephalin.

Acute and chronic pain (post-herpetic neuralgia or PHN) are encountered in patients with herpes zoster that is caused by reactivation of varicella-zoster virus (VZV) from a state of neuronal latency. PHN is often refractory to current treatments, and additional strategies for pain relief are needed. Here we exploited a rat footpad model of PHN to show that herpes simplex virus (HSV) vector-mediated gene delivery of human preproenkephalin (vHPPE) effectively reduced chronic VZV-induced nocifensive indicators of pain. VZV inoculated at the footpad induced prolonged mechanical allodynia and thermal hyperalgesia that did not develop in controls or with ultraviolet light-inactivated VZV. Subsequent footpad administration of vHPPE relieved VZV-induced pain behaviors in a dose-dependent manner for extended periods, and prophylactic vector administration prevented VZV-induced pain from developing. Short-term pain relief following low-dose vHPPE administration could be effectively prolonged by vector re-administration. HPPE transcripts were increased three- to fivefold in ipsilateral ganglia, but not in the contralateral dorsal root ganglia. VZV hypersensitivity and its relief by vHPPE were not affected by peripheral delivery of opioid receptor agonist or antagonist, suggesting that the efficacy was mediated at the ganglion and/or spinal cord level. These results support further development of ganglionic expression of enkephalin as a novel treatment for the pain associated with Zoster.

Further characterization of a rat model of varicella zoster virus-associated pain: Relationship between mechanical hypersensitivity and anxiety-related behavior, and the influence of analgesic drugs.

Persistent herpes zoster-associated pain is a significant clinical problem and an area of largely unmet therapeutic need. Progress in elucidating the underlying pathophysiology of zoster-associated pain and related co-morbidity behavior, in addition to appropriately targeted drug development has been hindered by the lack of an appropriate animal model. This study further characterizes a recently developed rat model of zoster-associated hypersensitivity and investigates (a) response to different viral strains; (b) relationship between viral inoculum concentration ('dose') and mechanical hypersensitivity ('response'); (c) attenuation of virus-associated mechanical hypersensitivity by clinically useful analgesic drugs; and (d) measurement of pain co-morbidity (anxiety-like behavior) and pharmacological intervention in the open field paradigm (in parallel with models of traumatic peripheral nerve injury). Varicella zoster virus was propagated on fibroblast cells before s.c. injection into the glabrous footpad of the left hind limb of adult male Wistar rats. Control animals received injection of uninfected fibroblast cells. Hind-limb reflex withdrawal thresholds to mechanical, noxious thermal and cooling stimuli were recorded at specified intervals post-infection. Infection with all viral strains was associated with a dose-dependent mechanical hypersensitivity but not a thermal or cool hypersensitivity. Systemic treatment with i.p. morphine (2.5 mg/kg), amitriptyline (10 mg/kg), gabapentin (30 mg/kg), (S)-(+)-ibuprofen (20 mg/kg) and the cannabinoid WIN55,212-2 (2 mg/kg) but not the antiviral, acyclovir (50 mg/kg), was associated with a reversal of mechanical paw withdrawal thresholds. In the open field paradigm, virus-infected and nerve-injured animals demonstrated an anxiety-like pattern of ambulation (reduced entry into the central area of the open arena) which was positively correlated with mechanical hypersensitivity. This may reflect pain-related co-morbidity. Further, anxiety-like behavior was attenuated by acute i.p. administration of gabapentin (30 mg/kg) in nerve-injured, but not virus-infected animals. This model will prove useful in elucidating the pathophysiology of zoster-associated pain and provide a tool for pre-clinical screening of analgesic drugs.

Gene structure of chick lumican and identification of the first exon.

Three overlapping genomic clones to chick lumican were isolated and then characterized using restriction enzyme analyses, Southern blot analyses with cDNA probes, and by DNA sequencing. The results showed chick lumican gene to consist of 3 exons with a 2.9-kb first intron and a 4.2-kb second intron. Transcription initiation sites, identified by S1 nuclease experiments using genomic fragments containing exon 1 and by primer extension analysis of RNA, indicated the first exon to be 303 b. Two TATA sequences were 31 and 49 bases upstream of the first exon. The first exon contained all 5' untranslated sequence. The second exon was 896 b and contains 20 b of untranslated sequence, and codes for the start methionine to the end of the 10th leucine rich repeat. The third exon is 880 b and codes for the remainder of the core protein, and 724 b of untranslated 3' sequence. A 1-kb genomic fragment containing a portion of exon 1 and upstream sequence in a luciferase reporter sector showed specific promotor activity in the forward, but not the reverse direction when transfected into corneal fibroblasts. These results show the chick lumican gene to consist of three exons, and that regulatory elements are present within 1 kb upstream of the first exon.

Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein.

Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or [3H]DS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to DS-DNA was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to DS-DNA as compared to SS-DNA. The binding to DS-DNA was reduced by 77% in the presence of 0.5 M NaCl, while the binding to SS-DNA was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding to DS-DNA. Following heparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both DS-DNA and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase alpha primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.

Molecular basis for a geographic variation of varicella-zoster virus recognized by a peptide antibody.

A live attenuated varicella vaccine, derived from a Japanese isolate, is currently being widely used to modulate disease caused by varicella-zoster virus. Differentiation of the vaccine from wild-type isolates has been and will continue to be critical in the assessment of the vaccine in the United States. This has largely relied upon identification of characteristic DNA polymorphisms in the vaccine strain in the United States. This has largely relied upon identification of characteristic DNA polymorphisms in the vaccine strain. In this report, we describe the identification of a new sequence polymorphism, located in the N-terminal coding sequence of open reading frame (ORF) 10. This variation results in the synthesis of an ORF 10 protein that is differentially recognized by antibodies to an ORF 10 synthetic peptide. The variation appears to be completely restricted to Japanese strains, including that used for the live attenuated varicella vaccine. As such, this polymorphism and the antibodies that differentially recognize it could prove highly useful in the assessment of the Japanese vaccine in the United States.

Transcriptional mapping of varicella-zoster virus regulatory proteins.

Varicella-zoster virus (VZV) expresses four proteins that influence viral transcriptional events and that also are homologous to herpes simplex virus type 1 (HSV-1) immediate-early proteins. However, their transcription and the mechanisms by which it is regulated are not yet resolved. To identify the promoter regions, a precise knowledge of the initiation and termination of the encoded RNAs is first required. In this report, we summarize the complete and precise mapping of the RNA transcripts of two of these genes--those from open reading frames 4 and 63. In addition, several elements of their promoter regulatory regions have been identified and predicted. Structural and functional studies of the regulatory sequences suggest that these two VZV genes may be regulated in a fashion different from that of their HSV-1 counterparts.

Latency of varicella zoster virus; a persistently perplexing state.

Varicella zoster virus (VZV) is the herpesvirus which causes the childhood disease varicella, also known as chickenpox, and the adult disease herpes zoster, also known as shingles. These distinct diseases are separated by a lengthy period of latency, often lasting decades, in which the virus resides within the ganglia of the host. VZV latency and reactivation from it have, for the most part, been extraordinarily difficult to examine. This is due to the lack of a good animal model for the VZV latent state, the inability to experimentally reactivate VZV under any circumstances and the caveats and problems encountered in examining human ganglionic tissue. However, insights into features of the molecular events of VZV latency have been gleaned from its pathogenesis and from recent advances in molecular probing of human and animal ganglia. Evidence suggests that the latent VZV genome may express transcripts unlike those of closely related herpesviruses, and some evidence suggests an unusual site for the establishment of VZV latency. In this review, the current evidence for events occurring during the VZV latent state will be discussed, from a view of its pathogenesis as well as its molecular biology.

Isolation of human adenovirus type 5 variants resistant to the antiviral cidofovir.

PURPOSE: Cidofovir (S-HPMPC) is a potent broad-spectrum antiviral drug with potential clinical application against infections caused by human cytomegalovirus, herpes simplex virus, and adenovirus (AD). This study sought to determine whether variants of AD5 could be isolated in vitro that demonstrated increased resistance to this new antiviral drug. METHODS: Homogenous stocks of wild-type AD5 (ATCC strain VR-5) were generated from isolated plaques grown in A549 cells. The stocks subsequently were serially passaged in cells containing increasing levels (from 5 to 75 micrograms/ml) of cidofovir. The recovered virus either was passaged, titrated, or assayed for 50% inhibitory concentration (IC50) of cidofovir. RESULTS: Three independently isolated variants were obtained that demonstrated increased resistance to cidofovir. Viral resistance to the drug increased on stepwise passage in higher concentrations. Compared to the ATCC AD5 reference (IC50 = 6.2 micrograms/ml), stable cidofovir-resistant variants showed fivefold to eightfold resistance (AD5 RI IC50 = 36.5 micrograms/ml; AD5 R2 IC50 = 36.7 micrograms/ml; and AD5 R3 IC50 = 32.6 micrograms/ml; analysis of variance, P = 0.000001). However, a variable number of passages (1 to 13) at each concentration of cidofovir was performed to obtain robust infectious virus suitable for testing at the next higher concentration. All resistant virus isolates grew to levels of virus titer comparable to the parental virus and showed no apparent phenotypic changes in growth rates, plaque size, or efficiency of plaque formation. CONCLUSIONS: The successful isolation of AD5 variants in tissue culture resistant to cidofovir has important clinical implications with respect to the anticipated use of this antiviral drug in treating adenoviral ocular infections.

Use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens.

PURPOSE: To evaluate the application of polymerase chain reaction (PCR) methodology as a potential diagnostic tool for the detection of adenovirus DNA in ocular swab samples. METHODS: Oligonucleotides derived from the adenovirus hexon gene were used to amplify a 306-base pair (bp) product by PCR. Radiolabeled oligonucleotides derived from sequences within the amplified product were used as specific probes. Specificity was determined against DNA of 13 adenovirus serotypes (types 1 to 11, inclusive, and types 19 and 37) and from nonadenoviral DNAs. Limits of detection were determined by PCR amplification of known amounts of purified adenovirus serotype 2 DNA. The assay was tested on 107 ocular swab samples and correlated to results obtained from tissue culture and a commercial immunoassay (Adenoclone). RESULTS: The 306-bp PCR product was amplified from all adenovirus serotypes tested, but not from negative control DNAs. As little as 15 fg of adenovirus type 2 DNA could be detected by PCR and ethidium bromide stain. Using a simplified sample preparation procedure, 46 of 58 adenovirus culture-positive but Adenoclone-negative swabs were positive by PCR (79% sensitivity). All (11 of 11) Adenoclone-positive clinical eye swabs tested were positive by PCR (100% sensitivity). Only 1 of 38 nonadenoviral ocular swab samples was positive by PCR (97% specificity). CONCLUSIONS: PCR appeared to be highly suitable for the diagnosis of adenovirus in ocular swabs, offering important improvements in speed over tissue culture isolation and in sensitivity over immunoassay.

The antiviral resistance and replication of cidofovir-resistant adenovirus variants in the New Zealand White rabbit ocular model.

PURPOSE: To determine the antiviral resistance of three cidofovir (CDV)-resistant variants of adenovirus type 5 (Ad5) and their ability to replicate in the New Zealand White rabbit ocular model. METHODS: Rabbits were inoculated topically in both eyes with the CDV-resistant variants R1, R2, and R3, and the Ad5 parental strain. On day 1, rabbits from each virus inoculation were divided into two topical treatment groups: 0.5% CDV and PBS control. Treatment was administered twice daily in both eyes for 7 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Using viral outcome parameters, CDV resistance was determined for each virus by comparing each CDV-treated virus group to its respective PBS control, and altered pathogenesis was assessed by comparing viral replication in the PBS control groups of the Ad5 parent and the three resistant variants. RESULTS: Topical 0.5% CDV treatment demonstrated significant antiviral inhibitory activity in the Ad5 parental group (e.g., reduced total Ad5-positive cultures, reduced daily Ad5-positive cultures on days 5, 9, 11, and 14, and duration of ocular shedding), but had no effect on the three CDV-resistant variants. There were no significant differences in pathogenicity between the Ad5 parent and the CDV-resistant variants. CONCLUSIONS: The Ad5 variants R1, R2, and R3 were resistant to topical treatment with 0.5% cidofovir in the rabbit ocular model. However, the acquisition of CDV resistance did not alter the replication of the three Ad5 CDV variants on the rabbit eye.

A Rho-associated protein kinase: differentially distributed in limbal and corneal epithelia.

PURPOSE: The authors have developed monoclonal antibodies (mAbs) to characterize the sequential biochemical changes in corneal epithelial cells after they differentiate from stem cells, located in the limbus, and migrate centripetally to follow the pathway of terminal differentiation. The purpose of this study was to identify a protein (recognized by mAb HE1/11F) with increased expression associated with the transition of the limbal epithelium to corneal epithelium. METHODS: The distribution and identification of the protein(s) were performed using an indirect immunohistochemical staining technique and a western blot analysis, respectively. A rabbit corneal epithelial cDNA library, constructed in the Uni-Zap XR vector, was screened with mAb HE1/11F to select cDNA clones expressing polypeptide(s) recognized by this mAb. Additional overlapping cDNA clones were obtained from a primer extension cDNA library to determine the sequence of the complete open reading frame encoding the protein recognized by mAb HE1/11F. RESULTS: Rabbit corneal epithelium exhibited strong immunostaining with mAb HE1/11F, however, the limbal epithelial cells stained weakly. HE1/11F recognized 160-kDa (HEBM1) and 100-kDa (HEBM2) polypeptides in the corneal epithelial extracts. The amino acid sequence of the protein deduced from the nucleotide sequence of the cDNA exhibited a close homology to that of a RhoA (Ras-related small GTPase)-associated serine-threonine kinase (ROCK-I or Rho-associated coiled-coil kinase). A 160-kDa RhoA-binding polypeptide with a molecular mass similar to that of HEBM1 and ROCK-I was detected in the corneal epithelial extracts. These findings strongly suggested that HEBM1 was rabbit ROCK-I. The identity of HEBM1 was further confirmed from the reactivity of mAb HE1/11F with ROCK-I immunoprecipitated from rabbit corneal epithelial extracts using anti-ROCK-I antibodies. CONCLUSIONS: The increased expression of a protein identified as ROCK-I from cDNA analyses is associated with rabbit corneal epithelial differentiation and transition from the limbal to corneal surface. Therefore, a RhoA signaling pathway is likely to be associated with corneal epithelial differentiation (maturation). A close homology among the cDNA sequences of rabbit, mouse, rat, and human ROCK-I indicates that this RhoA-associated kinase is a well-conserved protein.

The synthesis and immunogenicity of varicella-zoster virus glycoprotein E and immediate-early protein (IE62) expressed in recombinant herpes simplex virus-1.

In order to evaluate the conditions for optimal expression and immunogenicity of varicella-zoster virus (VZV) proteins in a herpes simplex virus-1 (HSV-1) vector, we selected the VZV glycoprotein E (gE), encoded by ORF 68 and the VZV product of ORF 62, an immediate-early major tegument protein (IE62). Three HSV/VZV recombinants were generated: (1) VZV gE protein coding sequences along with the promoter region were inserted into the thymidine kinase (TK) gene of HSV-1 strain KOS; (2) VZV gE expressed from the HSV-1 ICP4 promoter was inserted into the glycoprotein C (gC) gene of HSV-1 strain F; and (3) VZV IE62 protein coding sequences under the control of the HSV-1 ICP4 promoter were inserted into the gC gene of HSV-1 strain F. Immunoblot analysis and immunoperoxidase staining of infected cell monolayers demonstrated vector expression of VZV proteins. Following intracranial inoculation in mice, both VZV gE-HSV (TK) and VZV IE62-HSV (gC) induced an IgG response against VZV gE or VZV IE62. When tested in cytotoxicity assays using T-lymphocytes from VZV immune human donors, the range of precursor frequencies for T-lymphocytes that recognized VZV gE or VZV IE62 was similar whether these proteins were expressed by HSV-1 or a vaccinia vector. These experiments demonstrate that HSV-1 is a competent vector for expression of these VZV proteins and support the feasibility of engineering a combined vaccine for these closely related alpha-herpesviruses.

Nuclear retinoic acid receptors in the lacrimal gland.

The lacrimal gland secretes and metabolizes retinoids and responds to retinoic acid in culture. Like other retinoid responsive organs it is expected to express the nuclear retinoid receptors. The goal of this study was to identify the retinoic acid receptors (RAR) in the lacrimal glands of rats, rabbits, and humans. Total RNA was prepared from whole lacrimal glands and rat lacrimal gland acinar cells grown in culture. RNA was subjected to Northern blot analysis and probed for the RAR alpha, RAR beta, and RAR gamma mRNAs. Nuclear extracts of rat and rabbit lacrimal glands were incubated with 3H-all-trans retinoic acid and analyzed by gel filtration chromatography. Western blots of the nuclear extracts were probed using monoclonal antibodies to RAR alpha and RAR beta. Rat lacrimal gland expresses RAR alpha mRNA with two transcripts (3.8 and 3.0 kb), a single RAR beta mRNA transcript (3.3 kb), and a single RAR gamma mRNA transcript (3.3 kb). Cultured rat lacrimal acinar cells also expressed the mRNA for all three RAR subtypes. Rabbit lacrimal glands express mRNAs for RAR alpha (3.7 and 2.9 kb) and RAR beta (3.2 kb) but RAR gamma mRNA is not detectable. Human lacrimal glands also express mRNA for RAR alpha (3.5 and 2.3 kb), RAR beta (3.4 kb) and RAR gamma (3.0 kb). Lacrimal gland nuclear extracts contain proteins in the 50 kDa range that specifically bind retinoic acid with Kd = 1.25 nM in rat lacrimal gland and 0.3 nM in rabbit. The monoclonal antibodies identified RAR alpha and RAR beta in both rat and rabbit lacrimal glands. The results of this study support a role for retinoids in maintaining the structure and function of the lacrimal gland. The presence of RARs suggests potential interactions of these receptors with other members of their superfamily, including androgen and thyroid receptors, which also may be involved in lacrimal function.

Regulated nuclear localization of the varicella-zoster virus major regulatory protein, IE62.

The major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), IE62, accumulates within the nucleus of transfected and VZV-infected cells. Data are presented to show that nuclear localization of IE62 is dependent upon charged amino acids mapping to residues 677-685 of the 1310 residue protein. Furthermore, coexpression of VZV open-reading frame (ORF) 66 with IE62 results in the accumulation of cytoplasmic forms of IE62, suggesting that the ORF 66 protein can override the IE62 nuclear localization signal. Consistent with this, cytoplasmic forms of IE62 were detected in VZV-infected cells at late but not immediate early times after infection. The ORF 66 protein, a putative protein kinase, did not affect nuclear localization of other proteins, and IE62 nuclear localization was not affected by coexpression with VZV ORF 47, another putative kinase. These results suggest that IE62 nuclear functions may be specifically regulated by the VZV ORF 66 protein.

The transcriptional regulatory proteins encoded by varicella-zoster virus open reading frames (ORFs) 4 and 63, but not ORF 61, are associated with purified virus particles.

Of the five varicella-zoster virus (VZV) open reading frames (ORFs) known to encode proteins which influence viral transcriptional events, two (ORFs 10 and 62) encode proteins associated with the tegument of virus particles, where they may function during the immediate-early events of infection. In this study, antibodies which recognize the products of the three additional VZV ORFs, ORFs 4, 61, and 63, were made and used to characterize their association with virus particles. ORF 4 encoded a 52-kDa polypeptide, and antibodies to ORF 63 reacted with polypeptides of 47 and 28 kDa. Antibodies to ORF 61 recognized heterogeneous polypeptides of 62 to 66 kDa in cells infected with a vaccinia virus recombinant expressing ORF 61 and in VZV-infected melanoma cells but reacted very weakly with polypeptides of VZV-infected human foreskin fibroblasts, suggesting that cell-specific factors were involved in ORF 61 protein accumulation. Analysis of virus particles purified from melanoma cells indicated that a 52-kDa polypeptide from ORF 4 and the 47-kDa polypeptide from ORF 63, but not any from ORF 61, were associated with virus particles. The virion proteins were likely components of the tegument, as they were not solubilized by treatment of virus with mild detergents and were completely resistant to trypsin digestion unless prior envelope solubilization was performed. The products of ORFs 4 and 63 were not found in purified VZV nucleocapsids. These results suggest that forms of the ORF 4- and ORF 63-encoded transcriptional regulatory proteins are also structural and may also have roles in the immediate-early events of infection.

Nuclear accumulation of IE62, the varicella-zoster virus (VZV) major transcriptional regulatory protein, is inhibited by phosphorylation mediated by the VZV open reading frame 66 protein kinase.

IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.

Anatomy of the varicella-zoster virus open-reading frame 4 promoter.

The regulation of varicella-zoster virus (VZV) gene expression is largely controlled at the transcriptional level by a few key viral proteins cooperating with one another and with cellular transcription factors. However, the mechanisms involved are largely unclear. To identify the sequences important for the transcriptional regulation of open-reading frame (ORF) 4, itself encoding a transcriptional regulator, a mutation analysis of the promoter was done. These studies identified an element between -69 and -59 (relative to the transcriptional start site), which was critical to the activity of the promoter upon stimulation by the VZV transactivator IE62 and by VZV infection. DNA-protein interaction studies revealed that VZV induced the binding of a specific protein complex to this element, which contained the ubiquitous transcription factor USF. ORF 4 is the second VZV gene (in addition to VZV ORF 29) in which USF binding plays a critical role in gene expression.

The UL37 protein of herpes simplex virus type 1 is associated with the tegument of purified virions.

The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1) phosphoprotein in infected cells. Analysis of isolated mature HSV virions and light particles revealed that the UL37 protein is a component of the virion. Detergent solubilization and protease digestion experiments suggest that the UL37 protein is part of the tegument structure. Indirect immunofluorescence experiments using HSV-1-infected cells and cells infected with a vaccinia recombinant virus that expresses the UL37 gene demonstrated that the UL37 protein is present in both the nucleus and cytoplasm of infected cells and that localization to the nucleus does not require additional HSV proteins.

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.