Interleukin-18 protects mice against acute herpes simplex virus type 1 infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-gamma), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-gamma release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-gamma production was found in uninfected mice administered IL-18. Although IFN-gamma has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor NG-monomethyl-L-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-gamma produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.     

Dimerization and activation of the herpes simplex virus type 1 protease

The quaternary state of the herpes simplex virus type 1 (HSV-1) protease has been analyzed in relation to its catalytic activity. The dependence of specific activity upon enzyme concentration indicated that association of the 27-kDa subunits strongly increased activity. Size-exclusion chromatography identified the association as a monomer-dimer equilibrium. Isolation of monomeric and dimeric species from a size-exclusion column followed by immediate assay identified the dimer as the active form of the enzyme. Activation of the protease by antichaotropic cosolvents correlated with changes in the monomer-dimer equilibrium. Thus, dimerization of the enzyme was enhanced in solvents containing glycerol or the anions citrate or phosphate. These are substances previously identified as activators of HSV-1 protease (Hall, D. L., and Darke, P. L. (1995) J. Biol. Chem. 270, 22697-22700). The relative potencies of these cosolvents as enzyme activators correlated with their efficiency in promoting dimerization. Under all solvent conditions examined, the dependence of specific activity upon enzyme concentration was consistent with a kinetic model in which only the dimer is active. Dissociation constants for the HSV-1 protease dimer determined with this model at 15 degrees C, pH 7.5, were 964 and 225 nM in 20% glycerol with 0.2 and 0.5 M citrate present, respectively. The activation of the HSV-1 protease by antichaotropic cosolvents was hereby shown to be similar in nature to the activation of the other well characterized herpesvirus protease, that from human cytomegalovirus.     

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-alpha from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-alpha+ and most were also IL-6(+). Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-alpha and/or IL-6. IL-4(+) cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2(+) and/or IFN-gamma+ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-alpha and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

Cellular protein interactions with herpes simplex virus type 1 oriS

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.     

Stromal keratitis induced by a unique clinical isolate of herpes simplex virus type 1

The antibody responses of 65 volunteers receiving an i.d. regimen (0.1 ml given at two sites on days 0, 3, 7 and 0.1 ml given at one site on days 30 and 90) were compared with the control group of 35 volunteers receiving the standard i.m. regimen. By day 14, seroconversion was observed in all vaccinees in both groups. Geometric Mean Titers remained higher than 0.5 IU/ml throughout the study period. At the end of the observation period on day 365, antibodies persisted in all subjects. The multisite i.d. PCEC regimen has been proved as immunogenic as the standard i.m. regimen. Both regimens were well tolerated. Thus, it would be the effective and cheapest available rabies post-exposure treatment using tissue culture vaccine.     

Induced extrusion of DNA from the capsid of herpes simplex virus type 1

DNA-filled capsids (C capsids) of herpes simplex virus type 1 were treated in vitro with guanidine-HCl (GuHCl) and analyzed for DNA loss by sucrose density gradient ultracentrifugation and electron microscopy. DNA was found to be lost quantitatively from virtually all capsids treated with GuHCl at concentrations of 0.5 M or higher, while 0.1 M GuHCl had little or no effect. DNA removal from 0.5 M GuHCl-treated capsids was effected without significant change in the capsid protein composition, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or in its structure, as judged by electron microscopy. Electron microscopic examination of capsids in the process of emptying showed that DNA was extruded from multiple, discrete sites which appeared to coincide with capsid vertices. DNA exited the capsid in the form of thick strands or fibers that varied in diameter from approximately 4 to 13 nm with preferred diameters of 7 and 11 nm. The fibers most probably correspond to multiple, laterally aligned DNA segments, as their diameters are nearly all greater than that of a single DNA double helix. The results suggest that GuHCl treatment promotes an alteration in the capsid pentons which allows DNA to escape locally. Hexons must be more resistant to this change, since DNA loss appears to be restricted to the pentons. The ability of GuHCl to cause loss of DNA from C capsids with no accompanying change in capsid morphology or protein composition suggests that penton sites may open transiently to permit DNA exist and then return to their original state.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Lactoferrin inhibits herpes simplex virus type 1 adsorption to Vero cells

This paper describes the ability of human and bovine lactoferrins (HLf; BLf), iron-binding proteins belonging to the non-immune defense system, to interfere with herpes simplex virus type 1 (HSV-1) infection. Since lactoferrins are known to bind to heparan sulphate proteoglycans and to low density lipoprotein receptor, which in turn act as binding sites for the initial interaction of HSV-1 with host cells, we tested the effect of these proteins on HSV-1 multiplication in Vero cells. Both HLf and BLf are found to be potent inhibitors of HSV-1 infection, the concentrations required to inhibit the vital cytopathic effect in Vero cells by 50% being 1.41 microM and 0.12 microM, respectively. HLf and BLf exerted their activity through the inhibition of adsorption of virions to the cells independently of their iron withholding property showing similar activity in the apo- and iron-saturated form. The binding of [35S]methionine-labelled HSV-1 particles to Vero cells was strongly inhibited when BLf was added during the attachment step. BLf interacts with both Vero cell surfaces and HSV-1 particles, suggesting that the hindrance of cellular receptors and/or of viral attachment proteins may be involved in its antiviral mechanism.     

Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1

The heptadecapeptide orphanin FQ or nociceptin (OFQ/N), the endogenous ligand for the orphan opioid receptor, has a complex pharmacology in mice, eliciting either an anti-opioid/hyperalgesic action or analgesia depending upon the dose and testing paradigm. Unlike mice, orphanin FQ/nociceptin fails to elicit hyperalgesia in the rat following intracerebroventricular injection. Both OFQ/N and a truncated version, OFQ/N(1-11), produce a robust analgesic response. OFQ/N analgesia is readily antagonized by the opioid antagonists naloxone or diprenorphine, despite their very poor affinity for the cloned orphan opioid receptor. Antisense studies revealed that probes targeting the second and third coding exon of the orphan clone significantly attenuate OFQ/N analgesia, while the exon 1 probe was inactive. These results indicate that OFQ/N elicits a naloxone-sensitive analgesia in rats similar to that previously reported in mice.     

Recurrent ascending myelitis: an unusual presentation of herpes simplex virus type 1 infection

We report on a healthy female with a unique relapsing transverse myelitis accompanied by herpes simplex virus type 1 (HSV-1) infection. Magnetic resonance imaging showed cord enlargement and increased signal intensity on T1-weighted image with gadolinium enhancement from T-4 to T-10 during the first attack and from C-1 to C-2 during the second episode. She was not diagnosed during the first attack. During the second episode, laboratory studies disclosed IgM and IgG antibodies to HSV at the outset with greater than fourfold increases in antibody levels in the serum and cerebrospinal fluid (CSF). Cells cultured from the CSF were positive for HSV-1 according to the immunofluorescence method. The presence of HVS-1 DNA in CSF was documented by polymerase chain reaction (PCR) technique. Acyclovir was given with a partial recovery. We anticipate that PCR assay of CSF will assist early diagnosis of herpetic central nervous system disorders.     

The herpes simplex virus type 1 helicase-primase. Analysis of helicase activity

The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.     

Distribution of herpes simplex virus type 1 and 2 genomes in the human spinal ganglia

Herpes simplex virus (HSV) is well known for its propensity to cause recurrent oral or genital mucosal infections in humans. HSV-1 is involved primarily in oral lesions, whereas HSV-2 is more frequently involved in genital lesions. Based on this, it is thought that HSV-1 may produce latent infections in trigeminal ganglia, and HSV-2 in the sacral ganglia. However the distribution pattern of latent HSV-1 and HSV-2 infections in spinal ganglia remains unknown. Using the polymerase chain reaction we detected latent herpes HSV-1 and HSV-2 in human spinal ganglia obtained from autopsy material. A pair of primers which were specific for a part of the HSV-1 and HSV-2 DNA polymerase domain were employed. HSV-1 and HSV-2 DNAs were detected in 11 of 40 (28%) and 15 of 40 (38%) cervical ganglia, respectively, 52 of 103 (50%) and 47 of 103 (46%) thoracic ganglia, 16 of 53 (30%) and 17 of 53 (32%) lumbar ganglia, and 3 of 20 (15%) and 3 of 20 (15%) sacral ganglia. These findings suggest that latent HSV-1 and HSV-2 infections have a widespread distribution from the cervical ganglia to sacral ganglia. Importantly this study demonstrated latent HSV-1 infection of both the lumbar and sacral ganglia for the first time.     

Incorporation of nuclear matrix attachment regions into the herpes simplex virus type 1 genome does not induce long-term expression of a foreign gene during latency

We report two cases of skull base metastasis from renal cell carcinoma. Case 1: A 55-year-old female presented with a skull base tumor located on the clivus. Partial removal of the tumor was performed via the transsphenoidal approach. Case 2: A 44-year-old male presented with a skull base tumor occupying the orbital, nasal, and paranasal cavities. The first operation, partial removal of the tumor, was performed via the transsphenoidal approach. Thereafter, the tumor regrowth and the patient's symptoms were progressive, so a second operation was performed via Le-Forte I craniotomy to prevent cranial nerve dysfunction and air way obstruction. The initial symptom of these two cases was abducens nerve paresis, which is caused by the tumor extending from the clivus to the cavernous sinus. The neuroradiological features were the destruction of skull base bone and angiographic tumor stain. Histopathological examination of the surgically resected specimens revealed clear cell carcinoma, and thereafter they were diagnosed as skull base metastasis from renal cell carcinoma. We emphasize the necessity of a thorough medical workup to enable speedy diagnosis of renal cell carcinoma in such cases.     

Cloning and expression of certain herpes simplex virus type 1 genes in Escherichia coli

Complete genes IE12, IE63, IE68, IE175, UL19, UL29 of herpes simplex virus type I or their fragments have been cloned in Escherichia coli cells. The peptides expressed were shown to be fused with cro-beta-galactosidase proteins. The recombinant proteins containing amino acid sequences of ICP4, ICP27 and ICP47, major capsid protein, and major DNA-binding protein react in immunoblotting with the anti-HSVI serum from hyperimmune rabbit. The recombinant proteins can be used for creation of diagnosticums and other scientific and practical purposes. The immunological properties of the recombinant proteins are being investigated.     

Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity

The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.     

Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.