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1.
J Virol ; 78(10): 5038-44, 2004 May.
Article in English | MEDLINE | ID: mdl-15113885

ABSTRACT

The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17DeltaPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.


Subject(s)
Herpesvirus 1, Human/physiology , Keratitis, Herpetic/virology , Viral Proteins/physiology , Virus Latency , Animals , Cornea/virology , DNA, Viral/analysis , Disease Models, Animal , Genotype , MicroRNAs , Phenotype , Rabbits , Trigeminal Ganglion , Virus Replication
2.
Antimicrob Agents Chemother ; 45(7): 2044-53, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11408221

ABSTRACT

Famciclovir (FCV) is efficacious in the treatment of acute herpes zoster and recurrent genital infections but has not been used to treat ocular herpes simplex virus (HSV) infections. We evaluated the efficacy of orally administered FCV in treating HSV-1 epithelial keratitis and determined its effects on the establishment of latency and subsequent reactivation. Rabbits were inoculated with HSV-1 strain 17 syn+ and treated twice daily with increasing concentrations of FCV (60 to 500 mg/kg of body weight). This resulted in a significant, dose-dependent improvement in keratitis scores, as well as prolonged survival. Regardless of the dose of drug used, all groups exhibited the high rates of spontaneous and induced reactivation characteristic of 17syn+. The efficacy of 250 mg of FCV per kg was also compared to topical treatment with 1% trifluorothymidine (TFT). Although TFT treatment was more effective at reducing eye disease, FCV-treated rabbits had a better survival rate. Real-time quantitative PCR analysis of rabbit trigeminal ganglia (TG) demonstrated that FCV significantly reduced the HSV-1 copy number compared to that after treatment with TFT or the placebo but not in a dose-dependent manner. In summary, oral FCV treatment significantly reduces the severity of corneal lesions, reduces the number of HSV-1 genomes in the TG, improves survival, and therefore may be beneficial in reducing the morbidity of HSV keratitis in the clinic.


Subject(s)
2-Aminopurine/therapeutic use , Antiviral Agents/therapeutic use , Corneal Diseases/drug therapy , Herpes Simplex/drug therapy , Virus Latency/drug effects , 2-Aminopurine/analogs & derivatives , 2-Aminopurine/pharmacology , Acute Disease , Administration, Oral , Administration, Topical , Animals , Antiviral Agents/pharmacology , Corneal Diseases/mortality , Corneal Diseases/virology , Disease Models, Animal , Dose-Response Relationship, Drug , Famciclovir , Herpes Simplex/mortality , Herpes Simplex/virology , Herpesvirus 1, Human/drug effects , Keratitis, Herpetic/drug therapy , Keratitis, Herpetic/mortality , Rabbits , Trifluridine/pharmacology , Trifluridine/therapeutic use , Trigeminal Ganglion/virology , Viral Load
3.
Med Hypotheses ; 56(3): 348-56, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11359358

ABSTRACT

Stress has been shown to modulate an individual's immune system through the release of certain signal molecules such as catecholamines, cytokines and glucocorticoids. These signal molecules can significantly alter the host immune system and leave it susceptible to a primary or recurrent viral infection. Focusing on herpes simplex virus types-1 and -2 as examples, the authors explain how stress-associated immunomodulation can influence the recurrence of herpes simplex viral infections. Specific signal molecules such as epinephrine, interleukin-6, cyclic adenosine monophosphate, glucocorticoids and prostaglandins are upregulated during episodes of acute and chronic stress and have been implicated as effectors of herpes simplex viral reactivation and recurrent disease. The authors suggest that the release of immunomodulating signal molecules due to stress can compromise the host's cellular immune response and trigger herpes simplex viral reactivation.


Subject(s)
Herpes Simplex/immunology , Herpes Simplex/psychology , Stress, Psychological/immunology , Animals , Humans , Models, Immunological , Models, Psychological , Simplexvirus/growth & development , Virus Activation
4.
Virus Genes ; 23(3): 273-80, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11778695

ABSTRACT

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (43 degrees C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated 3-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (SOD-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.


Subject(s)
Gene Expression , Herpesvirus 1, Human/genetics , Trigeminal Ganglion/metabolism , Virus Latency , Animals , Female , Gene Expression Profiling , Herpesvirus 1, Human/physiology , Hyperthermia, Induced , Mice , Mice, Inbred BALB C , Oligonucleotide Array Sequence Analysis , Virus Activation
5.
Jpn J Ophthalmol ; 43(4): 257-61, 1999.
Article in English | MEDLINE | ID: mdl-10482469

ABSTRACT

PURPOSE: To determine if lamellar keratoplasty in rabbits latently infected with herpes simplex virus type 1 (HSV-1) would stimulate graft recipients to shed virus and induce viral-specific corneal lesions. METHODS: Rabbits latently infected with HSV-1 received lamellar allografts in one eye from normal uninfected rabbits and the contralateral eyes served as unoperated controls. Normal rabbits received lamellar grafts from rabbits latently infected with HSV-1. For 1 week after surgery, slit-lamp examination and ocular swab sampling were performed daily to assess viral reactivation. RESULTS: The occurrence of positive swab cultures and corneal epithelial lesions after lamellar keratoplasty was significantly higher in operated eyes of latently infected rabbits when compared to the control eyes. Ocular shedding or recurrent lesions were not observed in the normal rabbits receiving corneal grafts from latently infected donors. CONCLUSIONS: These results indicated that lamellar keratoplasty induces HSV-1 shedding and recurrent epithelial lesions in the eyes of rabbits latently infected with HSV-1, which received lamellar grafts, but not in the eyes of normal rabbits given lamellar grafts from HSV-1 latently infected rabbits. It seems that the site of viral latency is not the anterior corneal stroma or the epithelium.


Subject(s)
Corneal Transplantation/adverse effects , Epithelium, Corneal/virology , Herpesvirus 1, Human/physiology , Keratitis, Herpetic/etiology , Virus Activation , Animals , Epithelium, Corneal/pathology , Herpesvirus 1, Human/isolation & purification , Keratitis, Herpetic/pathology , Rabbits , Recurrence , Tears/virology , Transplantation, Homologous , Virus Latency/physiology , Virus Shedding/physiology
6.
J Virol ; 73(1): 767-71, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9847385

ABSTRACT

The herpes simplex virus type 1 (HSV-1) LAT gene is the only viral gene abundantly transcribed during latency. LAT null mutants created with strains McKrae and 17syn+ are impaired for both in vivo spontaneous and in vivo-induced reactivation. Thus, LAT is essential for efficient in vivo-induced and spontaneous reactivation. Different investigators have studied two LAT mutants containing a StyI-StyI region deletion corresponding to LAT nucleotides 76 to 447. One mutant, dLAT371 (parent strain, McKrae), had parental high frequencies of spontaneous reactivation. In vivo-induced reactivation was not examined. The other mutant, 17DeltaSty (parent strain, 17syn+), had parental frequencies of in vitro reactivation following cocultivation of explanted ganglia but reduced frequencies of in vivo-induced reactivation. Spontaneous reactivation frequency was not reported for 17DeltaSty. These combined results suggested the possibility that in vivo spontaneous reactivation and in vivo-induced reactivation may map to different regions within the LAT domain. We now report that dLAT371 has in vivo-induced reactivation frequencies of the parent and that 17DeltaSty has reduced frequencies of in vivo spontaneous reactivation. Thus, dLAT371 demonstrated the parental phenotype for both in vivo spontaneous and -induced reactivation while the apparently identical 17DeltaSty was impaired for both in vivo spontaneous and -induced reactivation. These results suggest that one or more differences between the genetic backgrounds of McKrae and 17syn+ result in different in vivo reactivation phenotypes of otherwise identical deletion mutations and that McKrae may have compensating sequences sufficient to overcome the loss of the StyI-StyI region of the LAT transcript.


Subject(s)
Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/physiology , Virus Activation , Virus Latency/genetics , Animals , Base Pairing , Genes, Viral , Mutation , Phenotype , Rabbits
7.
Mol Vis ; 5: 36, 1999 Dec 20.
Article in English | MEDLINE | ID: mdl-10617773

ABSTRACT

PURPOSE: Lysophosphatidic acid induces neurite retraction; it is also present in tears and aqueous humor. We determined whether lysophosphatidic acid induces HSV-1 reactivation in latently infected rabbits and whether the nerve growth associated protein GAP-43 undergoes posttranslational modification during the course of HSV-1 infection. METHODS: Rabbits were infected with HSV-1 and acute infection was documented by slit lamp examination. Corneas of latently infected rabbits were treated with lysophosphatidic acid or lysophosphatidylserine (structurally similar but lacking biological potency). For application to the cornea, these compounds were impregnated into collagen shields, applied as topical drops, or iontophoresed. In another experiment, corneas of latently infected rabbits were either untreated or treated iontophoretically with lysophosphatidic acid, lysophosphatidylserine, or saline. Ocular swabs detected shedding of infectious virus. Western blot and immunoprecipitation identified GAP-43 in corneal extracts and densitometry of silver-stained isoelectric focusing gels measured changes in GAP-43 isoform abundance. RESULTS: Iontophoresis of lysophosphatidic acid induced HSV-1 shedding more frequently than lysophosphatidylserine or saline. Viral shedding induced by collagen shield and topical drop administration was low and not significantly different for lysophosphatidic acid and lysophosphatidylserine. Five discrete GAP-43 isoforms predominated in the IEF gels. Most abundant were the pI 4.7 band in uninfected cornea and the pI 5.05 band in latently-infected cornea. Compared to latently-infected cornea, there was no significant change in isoform abundance 1 h after lysophosphatidic acid iontophoresis, but 24 and 72 h later, the pI 5. 05 band was diminished. CONCLUSIONS: Lysophosphatidic acid can induce HSV-1 reactivation and changes in GAP-43 pI suggest that posttranslational modifications, possibly related to phosphorylation and ADP-ribosylation, are occurring during HSV-1 latency and after LPA is iontophoretically applied to the cornea. How lysophosphatidic acid-induced signaling, HSV-1 reactivation, and GAP-43 pI are related remains to be determined.


Subject(s)
GAP-43 Protein/metabolism , Herpesvirus 1, Human/growth & development , Lysophospholipids/administration & dosage , Virus Activation/drug effects , Animals , Herpesvirus 1, Human/drug effects , Iontophoresis , Isoelectric Point , Keratitis, Herpetic/metabolism , Keratitis, Herpetic/virology , Lysophospholipids/pharmacology , Protein Isoforms , Protein Processing, Post-Translational , Rabbits , Time Factors , Virus Shedding/drug effects
8.
J Virol ; 71(9): 7114-8, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9261446

ABSTRACT

The protease domain of the murine cytomegalovirus (MCMV) M80 open reading frame was expressed in and purified from Escherichia coli. The recombinant enzyme was recovered as a mixture of active one- and two-chain forms. The two-chain enzyme was formed by internal cleavage of the one-chain enzyme at the I site. Activity measurements showed that MCMV protease cleaves R- and M-site peptide mimics with kinetics similar to those of recombinant human cytomegalovirus (HCMV) protease. Both the MCMV and HCMV proteases cleave I-site peptide substrates very poorly, but the crystal structure of the HCMV protease indicates that the cytomegalovirus I site likely resides on a solvent-exposed loop close to the active site.


Subject(s)
Endopeptidases/metabolism , Muromegalovirus/enzymology , Amino Acid Sequence , Animals , Binding Sites , Endopeptidases/chemistry , Endopeptidases/genetics , Gene Expression , Humans , Mice , Molecular Sequence Data , Molecular Structure , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
9.
J Virol ; 71(9): 6555-9, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9261376

ABSTRACT

In this study we used a herpes simplex virus type 1 (HSV-1) deletion mutant to identify a segment of the genome necessary for epinephrine-induced reactivation in the rabbit eye model of herpetic recurrent disease. In HSV-1 latently infected neural tissue, the only abundant viral products are the latency-associated transcripts (LATs). At least one promoter of LAT has been identified, and mutations in the LAT domain have been used to investigate HSV-1 reactivation. We used an ocular rabbit model of epinephrine-induced HSV-1 reactivation to study the effects of deleting a 437-bp region beginning 796 bp upstream of the LAT CAP site. Specifically, the 437-bp deletion is located between genomic positions 118006 and 118443 of the parent 17Syn+, and the construct is designated 17 delta S/N. This region also controls a portion of the genome encoding two transcripts (1.1 and 1.8 kb) from the LAT domain. A rescuant, 17 delta S/N-Res, was constructed from 17 delta S/N. Following ocular infection, all three viruses produced similar acute dendritic lesions in rabbits. Five weeks after infection, rabbits received transcorneal iontophoresis of epinephrine. The parent, 17Syn+, and the rescuant, 17 delta S/N-Res, underwent a high frequency of HSV-1 ocular reactivation as determined by recovery of infectious virus in the tear film. Rabbits infected with 17 delta S/N had a significantly lower frequency of ocular reactivation. Analysis of the trigeminal ganglia from all three groups of latently infected rabbits revealed (i) similar amounts of HSV DNA (genomic equivalents), (ii) accumulation of 2.0- and 1.45-kb LATs, and (iii) explant reactivation at the same high frequency. Therefore, these studies indicate that the 437-bp deleted region in 17 delta S/N is essential for epinephrine-induced reactivation and could implicate the 1.1- and 1.8-kb transcripts in the mechanisms controlling HSV-1 reactivation.


Subject(s)
Herpesvirus 1, Human/genetics , Keratitis, Herpetic/virology , Promoter Regions, Genetic , Virus Activation/genetics , Adrenergic Agonists/pharmacology , Animals , Base Composition , Blotting, Southern , Cell Line , Chlorocebus aethiops , Cornea/virology , DNA, Viral/metabolism , Epinephrine/pharmacology , Genotype , Humans , Rabbits , Sequence Deletion , Trigeminal Ganglion/virology , Vero Cells , Virus Activation/drug effects , Virus Latency
10.
Biochem Biophys Res Commun ; 203(1): 472-8, 1994 Aug 30.
Article in English | MEDLINE | ID: mdl-8074692

ABSTRACT

The murine cytomegalovirus UL80 open reading frame was cloned and the predicted amino acid sequence compared with those from other herpesviruses. The open reading frame encodes a fused protease-capsid assembly protein precursor and maintains conserved features including the active site serine, conserved regions CD1 through CD5, the release and maturation sites, and a potential internal cleavage site within the protease. However, the murine cytomegalovirus protease differs in comparison with the other proteases because it contains a unique 15-16 amino acid insertion between CD3 and CD1. The assembly protein sequences are relatively divergent, but they can be arranged into groups defined by herpesvirus subfamily, with each group possessing a conserved motif at its carboxyl terminus.


Subject(s)
Capsid/genetics , Cytomegalovirus/genetics , Endopeptidases/genetics , Genes, Viral , Amino Acid Sequence , Animals , Base Sequence , Capsid/biosynthesis , Cloning, Molecular , Conserved Sequence , Endopeptidases/biosynthesis , Herpesviridae/genetics , Mice , Molecular Sequence Data , Open Reading Frames , RNA, Viral/metabolism , Sequence Homology, Amino Acid
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