Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.     

Regulated high-level expression of the Herpes simplex type I thymidine kinase gene in Escherichia coli

A plasmid-borne Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene (tk) was expressed in Escherichia coli by inserting a 203-bp lacL8/UV5 promoter-operator segment, in frame, 53 bp 5' to the native tk translational start codon. The hybrid gene created by this fusion encodes a polypeptide which has 25 additional amino acids on the amino terminus of the HSV-1 TK protein and phenotypically complements a tdk- mutation of E. coli. This fusion polypeptide has been characterized by maxicell, immunoprecipitation, and native gel techniques, and its activity is inhibited by anti-HSV-1 antibody. In a tk expressor strain containing a F' lacIq (which overproduces the lactose repressor), the isopropyl-beta-D-thiogalactoside (IPTG) causes greater than 1000-fold coordinate induction of the plasmid-encoded TK and chromosomal beta-galactosidase activities. Pulse-chase induction demonstrates the fused TK polypeptide to be as stable as beta-galactosidase. HSV-1 tk-specific RNA isolated from this bacterial strain has a short half-life characteristic of bacterial messages.     

Immunogenic activity of temperature sensitive mutation of herpes simplex virus type-1 in mono- and polyvalent conditions with different attenuated strains of virus. I. Analysis of humoral immunity induced by mutant temperature sensitive herpes simplex virus type-1 in monovalent systems

The aim of the present study was the analysis of the immunogenic activity of temperature sensitive mutant of HSV-1 with significantly decreased pathogenicity for mice. The stability of immunogenic potency was also tested. The level of antibody-mediated response in guinea pigs after two doses of mutant ts HSV-1 or native strain was compared. Antibodies to HSV were measured using ELISA and CFM. Seroconversion rates in tested groups were high, from 80% to 100%. The level of antibody response at 4 week after inoculation measured by ELISA and CFM showed no significant differences between 4 passages of mutant ts HSV-1. Significant higher increase of antibodies level after two doses of viruses was observed for mutant ts HSV-1 as compared to native strain, both in ELISA and CFM.     

Immunogenic activity of HSV-1 temperature sensitive mutant's proteins in mono- and polyvalent systems of immunization

Immunogenic activity of herpes simplex type 1 temperature sensitive mutant's (ts HSV-1 mutant) proteins was tested in two systems: monovalent and polyvalent with other attenuated virus strains (measles and mumps). The guinea pigs were used as animal model. In monovalent system the humoral response in animals infected with ts HSV-1 mutant (1 or 2 doses) was studied and compared to results received for HSV-1 native strain. In polyvalent system the immunological response induced by ts HSV-1 mutant in the presence of RNA virus strains was tested.     

Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.     

The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme.

The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products. A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability. The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase. The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM. The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration. In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+. The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine. Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA. Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient. However, the enzymes differ immunologically.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.     

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.     

Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome

Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.     

4'-alpha-C-Branched N,O-nucleosides: synthesis and biological properties

The synthesis of 4'-alpha-C-branched N,O-nucleosides has been described, based on the 1,3-dipolar cycloaddition of nitrones with vinyl acetate followed by coupling with silylated nucleobases, The obtained compounds have been evaluated for their activity against HSV-1, HSV-2, HTLV-1. Cytotoxicity and apoptotic activity have been also investigated: compound 10c shows moderate apoptotic activity in Molt-3 cells.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.     

Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate that an HSV-1 amplicon expressing the AAV-2 genes rep and cap along with HSV-1 helper functions supports the replication and packaging of rAAV vectors in a scaleable process.     

Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells

Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.     

Inhibition of Herpes Simplex Virus-1 Replication by Natural Compound Honokiol

Honokiol is a pleiotropic natural compound isolated from Magnolia and has multiple biological and clinically relevant effects, including anticancer and antimicrobial function. However, the antiviral activity of honokiol has not yet been well studied. Here we showed that honokiol had no effect on herpes simplex virus-1(HSV-1) entry, but inhibited HSV-1 viral DNA replication, gene expression and the production of new progeny viruses. The combination of honokiol and clinical drug acyclovir augmented inhibition of HSV-1 infection. Our results illustrate that honokiol could be a potential new candidate for clinical consideration in the treatment of HSV-1 infection alone or combination with other therapeutics.     

Physical mapping of the herpes simplex virus type 2 nuc- lesion affecting alkaline exonuclease activity by using herpes simplex virus type 1 deletion clones

The nuc- lesion affecting alkaline exonuclease activity in the herpes simplex virus type 2 (HSV-2) mutant ts1348 had previously been mapped to the EcoRI-D restriction enzyme fragment of HSV-1. Eight clones with deletions representing most of HSV-1 EcoRI fragment D were selected with lambda gtWES hybrids. These clones were tested for their ability to rescue the alkaline exonuclease activity of HSV-2 nuc- ts1348 virus. The sequences colinear with the HSV-2 nuc- lesion were found to map between 0.169 and 0.174 map units on the HSV-1 Patton genome, representing an 0.8-kilobase-pair region that is 12.9 to 13.7 kilobase pairs from the left end of HSV-1 EcoRI fragment D.     

Cell-free assembly of the herpes simplex virus capsid.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.     

Synthesis of 2',3'-dideoxy-2'-fluoro-L-threo-pentofuranosyl nucleosides as potential antiviral agents

A series of 2',3'-dideoxy-2'-fluoro-L-threo-pentofuranosyl nucleosides has been synthesized as potential antiviral agents. The synthesized compounds were evaluated against HIV-1, HBV, HSV-1, and HSV-2. Among the synthesized analogues, only the cytosine derivative showed moderate antiviral activity against HIV and HBV.     

Site-Directed Mutagenesis of the Virion Host Shutoff Gene (UL41) of Herpes Simplex Virus (HSV): Analysis of Functional Differences between HSV Type 1 (HSV-1) and HSV-2 Alleles

During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157-7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.