Genetic and functional complementation of the HSV1 UL27 gene and gB glycoprotein by simian alpha-herpesvirus homologs.

Utilizing co-transfection of DNA from glycoprotein gB- strain of HSV1 and cloned fragments of several simian alpha-herpesviruses containing the UL26, UL27 (gB glycoprotein), and UL28 gene homologs, replication-competent recombinant viruses were produced. Genetic analysis of one HSV1/SA8 recombinant (HSV1/SgB) demonstrated the presence of SA8 DNA comprising the entire UL27 (gB) gene and parts of the UL28 and UL26 ORFs in an otherwise HSV1 genome. The recombinant was shown to express the SA8 gB and p40 proteins (UL27 & UL26.5 gene products, respectively); all other proteins were indistinguishable from those of HSV1. The recombinant behaved like SA8 in gB-specific virus neutralization and cell surface antibody binding assays, while plaque morphology and replication kinetics were very similar to HSV1. Despite its overwhelming HSV1 genetic constitution, the recombinant displayed a pathogenic phenotype in mice very different from the parental HSV1. While HSV1 produced corneal disease in ocularly infected mice and readily spread to the nervous system. HSV1/SgB was markedly impaired in both respects. These results demonstrate the functional equivalency of the cercopithecine monkey virus gB glycoproteins and genes (including transcriptional regulatory elements) in HSV1, the functional nature of HSV1/SA8 chimeric UL28 and UL26 genes/proteins, and that UL28, gB and/or p40 proteins may effect the pathogenicity of HSV1.

Characterization of an essential HSV-1 protein encoded by the UL25 gene reported to be involved in virus penetration and capsid assembly.

The 2.3-kb BamHI-U DNA fragment (map units 0.319-0.335) of herpes simplex virus type 1 (HSV-1) genome contains the complete UL25 open reading frame (ORF). It specifies an essential viral protein reported previously to be involved in virus penetration and capsid assembly (C. Addison, F. J. Rixon, J. W. Palfreyman, M. O'Hara, and V. G. Preston, Virology 138, 246-259, 1984). To identify the protein encoded by the UL25 gene, the UL25 ORF was cloned in a eukaryotic expression vector (p91023) downstream of the adenovirus major late promoter to generate the expression plasmid p9-UL25. Synthesis of a 60-kDa protein was observed in COS-7 cells transfected with p9-UL25 plasmid DNA, but not in cells transfected with p91023 control plasmid DNA. To identify and characterize the UL25 protein from HSV-1-infected cells, we prepared a rabbit antiserum by using UL25-GST fusion protein expressed in Escherichia coli as immunogen. This rabbit antiserum readily immunoprecipitated the 60-kDa UL25 protein from HSV-1-infected cells. In HSV-1-infected cells, UL25 protein was expressed as a late (gamma) or a leaky late (gamma 1) viral protein. The rabbit antiserum raised against HSV-1 UL25 protein immunoprecipitated a UL25-homologue of identical size from HSV-2-infected cells. However, the reactivity of the antiserum with HSV-2 UL25-homologue was weaker than compared to the corresponding HSV-1 protein. Consistent with its classification as a virion component, the UL25 protein was found to be associated with purified HSV-1 virions.

Deletion of the carboxy-terminus of herpes simplex virus type 1 (HSV-1) glycoprotein B does not affect oligomerization, heparin-binding activity, or its ability to protect against HSV challenge.

A recombinant vaccinia virus designated VgBt which expresses a truncated secreted herpes simplex virus gB (gBt) was constructed and compared to V11gB, a vaccinia recombinant previously studied which expresses gB exclusively on the surface of infected cells. Indirect immunofluorescence assay (IFA) revealed that gBt was strongly associated with the surface of infected cells despite being released slowly into the cell culture medium. Both gB and gBt existed as oligomers, and both membrane bound and secreted forms of gBt exhibited heparin-binding activity. In protection studies VgBt and V11gB conferred equivalent protection against both homologous (HSV-1) and heterologous (HSV-2) challenge with HSV.

Gamma interferon expression during acute and latent nervous system infection by herpes simplex virus type 1.

This study was initiated to evaluate a role for gamma interferon (IFN-gamma) in herpes simplex virus type 1 (HSV-1) infection. At the acute stage of infection in mice, HSV-1 replication in trigeminal ganglia and brain stem tissue was modestly but consistently enhanced in mice from which IFN-gamma was by ablated monoclonal antibody treatment and in mice genetically lacking the IFN-gamma receptor (Rgko mice). As determined by reverse transcriptase PCR, IFN-gamma and tumor necrosis factor alpha transcripts were present in trigeminal ganglia during both acute and latent HSV-1 infection. CD4+ and CD8+ T cells were detected initially in trigeminal ganglia at day 5 after HSV-1 inoculation, and these cells persisted for 6 months into latency. The T cells were focused around morphologically normal neurons that showed no signs of active infection, but many of which expressed HSV-1 latency-associated transcripts. Secreted IFN-gamma was present up to 6 months into latency in areas of the T-cell infiltration. By 9 months into latency, both the T-cell infiltrate and IFN-gamma expression had cleared, although there remained a slight increase in macrophage levels in trigeminal ganglia. In HSV-1-infected brain stem tissue, T cells and IFN-gamma expression were present at 1 month but were gone by 6 months after infection. Our hypothesis is that the persistence of T cells and the sustained IFN-gamma expression occur in response to an HSV-1 antigen(s) in the nervous system. This hypothesis is consistent with a new model of HSV-1 latency which suggests that limited HSV-1 antigen expression occurs during latency (M. Kosz-Vnenchak, J. Jacobson, D.M. Coen, and D.M. Knipe, J. Virol. 67:5383-5393, 1993). We speculate that prolonged secretion of IFN-gamma during latency may modulate a reactivated HSV-1 infection.

Herpes simplex virus DNA in normal corneas: persistence without viral shedding from ganglia.

Herpes simplex virus type 1 (HSV-1) DNA has been shown to persist in the cornea not only after inoculation of experimental animals but also in surgical samples from patients with herpes keratitis. The further observation of corneal HSV-1 DNA in subjects without known HSV eye disease prompted the present study of the presence and distribution of HSV-1 in eye bank corneas. Prior to DNA extraction, the corneas were trephined, separating the central and peripheral cornea. With polymerase chain reaction (PCR) for HSV-1 thymidine kinase (TK) and glycoprotein D (gD) gene sequences, we found HSV-1 in 10 of 24 eye bank corneas, from the 4 mm wide corneal rim in 8 eyes and from the 8 mm diameter central cornea in 2 eyes. In 9 subjects, both eyes were assayed, and HSV-1 was detected in 6 subjects. In only one subject was HSV-1 detected in both eyes and in only one subject was HSV-1 detected in the central and peripheral cornea of the same eye. The biological role of HSV-1 DNA corneal sequences is unknown. To investigate this, a rabbit animal model was established by transplantation of corneas containing viral DNA sequences in HSV-1 naive recipients. Followed for 5 months, there was no evidence of sheeding of HSV-1 in the tear film or seroconversion of the recipient rabbits. At the end of this time, HSV-1 DNA was detected in the corneal graft at a similar intensity to the PCR signal from the donor rims.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo effects of synthetic ribozymes targeted against BCR/ABL mRNA.

Chronic myelogenous leukemia (CML) is associated with a translocation of the BCR and the ABL genes, t(9;22). Results of this event are transcription and translation products that are unique to malignant cells. We therefore designed synthetic ribozymes which are capable of exclusively cleaving the BCR/ABL B3A2-type mRNA without altering normal cellular transcripts. Synthetic B3A2-type transcripts could only be cleaved by B3A2-type mRNA targeted ribozymes and not by any of the controls. The B3A2-type mRNA directed ribozyme, on the other hand, did not cleave any of the control transcripts. The effective delivery of ribonucleic acids by lipofection into K562 cells could be demonstrated by fluorescent microscopy, slot blot analysis, and RNA polyacrylamide gel electrophoresis. In vivo, we were able to induce a significant inhibition of the proliferation of K562 cells with ribozymes directed against the B3A2-type mRNA. Quantitative PCR analyses showed an up to fivefold reduction of the relative number of BCR/ABL mRNA molecules per single cell after exposure to ribozymes compared to controls. We conclude that ribozymes targeted against the B3A2-type BCR/ABL mRNA function in vitro and in vivo.

Antisense oligonucleotides as antiviral agents: prospects and problems.

One possible strategy for the development of antiviral drugs is to synthesize short antisense oligonucleotides that interfere specifically with RNA transcription and processing to prevent expression of protein. This can be readily achieved, but there are formidable technical problems to solve before routine clinical applications can be envisaged.

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

Application of polymerase chain reaction assays to studies of herpes simplex virus latency.

We have amplified herpes simplex virus type 1 (HSV-1) DNA sequences from individual latently infected mouse trigeminal ganglia by polymerase chain reaction (PCR) assays. This report presents two useful modifications in the PCR technique. The first involves the use of two sets of closely spaced, oppositely oriented oligonucleotide primers and two rounds of 20-40 PCR cycles, first with the more widely spaced outer primers and then with the internal nested primers. This method enhanced the sensitivity of PCR detection as shown by assays of HSV-1 sequences in human brain. The second modification was designed to detect selectively HSV-1 sense or anti-sense RNA transcripts when both are present by adding a single primer during an initial reverse-transcriptase-mediated cDNA synthesis reaction. After destruction of the RNA template, standard PCR is initiated by the addition of the second primer and thermus aquaticus DNA polymerase (Taq). We show here applications of both of these modifications to amplify HSV-1 sequences from nervous system tissue.

The potential use of catalytic RNAs in therapy of HIV infection and other diseases.

This article describes the applications (both real and potential) of a new antiviral strategy, based on the use of antisense, catalytic RNAs (ribozymes) as therapeutic agents. An understanding of both antisense inhibition of gene expression and RNA autocleavage reactions are essential to the use of this technology. In addition, for the successful application of this technology in clinical settings, an interdisciplinary approach involving clinicians, molecular and cellular biologists, will be necessary. The following treatise will highlight several salient features of ribozyme technology, emphasizing its application as an antiviral as well as discuss some problems and potential solutions pertinent to the clinical application of this technology.

Detection of herpes simplex virus DNA sequences in corneal transplant recipients by polymerase chain reaction assays.

Polymerase chain reaction (PCR) assays were used to amplify herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) sequences in DNA extracted from formalin-fixed, paraffin embedded corneas of patients undergoing corneal transplantation. PCR reamplification with an internal (nested) set of primers was required for detection in 10 of the 12 positive corneas indicating very low abundance of viral sequences. Three of the positive corneal samples were from failed corneal grafts. Overall, TK sequences were detected in 8 of 11 corneas from subjects with a past history of herpes keratitis and in 4 of 11 corneas from subjects with no past history of herpetic eye disease.

Ribozymes as potential anti-HIV-1 therapeutic agents.

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.

Inhibition of human immunodeficiency virus by using an oligonucleoside methylphosphonate targeted to the tat-3 gene.

Antiviral effects were characterized for two oligodeoxyribonucleoside methylphosphonates synthesized in an antisense (3'-TCTTAACC-5') or a sense (5'-AGAATTGG-3') orientation, based on the RNA sequence of the first splice acceptor site of the tat-3 gene of human immunodeficiency virus (HIV) (5'...AGAAUUGG...3'). The development of syncytial cells and supernatant reverse transcriptase was inhibited by a single exposure to the antisense HIV, and HIV RNA synthesis was inhibited by both antisense and sense methylphosphonates but not by a control herpes simplex virus antisense sequence.

A recombinant vaccinia virus expressing herpes simplex virus type 1 glycoprotein B induces cytotoxic T lymphocytes in mice.

Spleen cells from BALB/c (H-2d) mice vaccinated with vgB11, a recombinant vaccinia virus which expresses glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), lysed EMT6 (H-2d) target cells infected with vgB11 or with HSV-1 but did not lyse uninfected EMT6 cells or infected L-929 (H-2k) target cells. Unlabelled target cell competition of lysis showed that only syngeneic cells infected with vgB11 or HSV-1 inhibited lysis of radiolabelled HSV-1-infected targets. These results demonstrate that vgB11 induces H-2-restricted anti-HSV-1 cytotoxic T lymphocytes and that gB is the target antigen.

Monoclonal antibodies to HSV-infection-related antigens cross-react with tumor cell lines and tumor tissue sections.

The purpose of our study was to evaluate a possible association between herpes simplex virus (HSV) and various tumors, including oral squamous cell carcinoma (SCC). To this end, we tested the binding of appropriate monoclonal antibodies to a panel of cell lines and tumor sections. The 25 monoclonal antibodies were reactive with HSV-infected cells but not with uninfected cells. Of these antibodies, three bound to several SCC cell lines and to one non-SCC cell line (K562). One of these three antibodies also reacted with sections of oral SCC tumors, the adjacent mucosa, and normal esophageal epithelium. In contrast, it did not bind to sections of kidney, spleen, esophageal smooth muscle, and skin. To evaluate whether the observed antibody binding could reflect an actual infection by HSV, hybridization experiments between K562 DNA and HSV DNA were performed. HSV DNA sequences were found in K562 DNA at a ratio of 0.1 genome-equivalents/cell. Although these data do not characterize the nature of the relationship, they clearly confirm the postulated association of HSV with some tumors (in particular oral SCC).

Expression of herpes simplex virus 1 glycoprotein B by a recombinant vaccinia virus and protection of mice against lethal herpes simplex virus 1 infection.

The herpes simplex virus 1 (HSV-1) strain F gene encoding glycoprotein gB was isolated and modified at the 5' end by in vitro oligonucleotide-directed mutagenesis. The modified gB gene was inserted into the vaccinia virus genome and expressed under the control of a vaccinia virus promoter. The mature gB glycoprotein produced by the vaccinia virus recombinant was glycosylated, was expressed at the cell surface, and was indistinguishable from authentic HSV-1 gB in terms of electrophoretic mobility. Mice immunized intradermally with the recombinant vaccinia virus produced gB-specific neutralizing antibodies and were resistant to a lethal HSV-1 challenge.

The immediate-early enhancer element of herpes simplex virus type 1 can replace a regulatory region of the c-Ha-ras1 oncogene required for transformation.

A 0.8-kilobase SacI DNA fragment in the distal 5'-noncoding region of the c-Ha-ras1 oncogene hybridized to high guanine X cytosine sites of herpes simplex virus type 1 (HSV-1) DNA restriction fragments. Nucleotide sequence comparisons localized one of these sites to the intergenic region of HSV between the immediate-early genes coding for IEmRNA-3 and IEmRNA-4/5 that has enhancer-type activity. We tested the possibility that the HSV-1 enhancer and the upstream c-Ha-ras1 SacI fragment were functionally related by assaying for the capacity of recombinant plasmids in which the HSV-1 enhancer replaced the oncogene 0.8-kilobase SacI fragment to transform NIH/3T3 cells. Deletion of the 0.8-kilobase SacI fragment abolished the biological activity of c-Ha-ras1, but its replacement by the HSV-1 enhancer fully restored it. These results confirm the enhancer properties of the HSV-1 immediate-early intergenic region and suggest that c-Ha-ras1 sequences contained within the 0.8-kilobase SacI fragment plays a role in the transcriptional activation of the oncogene.