Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Structure of the joint region and the termini of the DNA of herpes simplex virus type 1.

Analysis of restriction endonuclease cleavage sites within the inverted, repeated sequences in the joint region of the DNA of herpes simplex virus type 1 strain KOS revealed the presence of two types of sequence heterogeneity. The first was an insertion of 280 base pairs or multiples of 280 base pairs which was found in approximately half of all DNA molecules from every plaque-purified stock of virus. These insertions seemed to be tandem duplications of sequences which were present at the joint and correspond closely to the inverted terminal redunancy. The second type of heterogeneity was due to variable insertions and deletions which were present in some, but not all, plaque-purified virus stocks. Comparison of restriction fragments from the joint region with fragments from the termini indicated that in the simplest observed molecules of herpes simplex virus type 1 DNA, only one copy of the inverted terminal redundancy was present at the joint. A map of restriction endonuclease cleavage sites in the joint region is presented.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.     

Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay.

Two mutations affecting herpes simplex virus type 1 glycoprotein B were mapped by marker rescue using cloned sequences of wild-type herpes simplex virus type 1 strain KOS DNA. One mutant, tsB5, is a temperature-sensitive mutant which does not express mature, functional glycoprotein B at the nonpermissive temperature. The other mutant, marB1.1, expresses an antigenic variant of glycoprotein B and was selected for resistance to neutralization by a monoclonal antibody. The mutation in tsB5 mapped to a 1.2-kilobase segment of the herpes simplex virus type 1 genome between coordinates 0.361 and 0.368, whereas the mutation in marB1.1 mapped to a 1.6-kilobase segment between coordinates 0.350 and 0.361. An in situ enzyme immunoassay was used to detect plaques of recombinant wild-type virus among the progeny of transfections with mutant marB1.1 DNA and wild-type DNA fragments.     

Use of monoclonal antibodies against two 75,000-molecular-weight glycoproteins specified by herpes simplex virus type 2 in glycoprotein identification and gene mapping

We produced two monoclonal antibodies that precipitate different glycoproteins of similar apparent molecular weight (70,000 to 80,000) from extracts of cells infected with herpes simplex virus type 2. Evidence is presented that one of these glycoproteins is the previously characterized glycoprotein gE, whereas the other maps to a region of the herpes simplex virus type 2 genome collinear with the region in herpes simplex virus type 1 DNA that encodes gC.     

Detection of herpes simplex virus by DNA-DNA hybridization method]

Eight recombinant clones were obtained by insertion of BamHI fragments of herpes simplex type I viral DNA into a vector plasmid pUC19o. Of the obtained clones 5 were found to hybridize with herpes simplex type I and 2 viral DNA while 3 clones revealed a positive reaction with the Vero cells DNA. A constructed DNA-probe possessing the highest level of activity was selected for further studies. The probe is a BamHI fragment of herpes simplex type I viral DNA labelled with 32P dTTP. Probe sensitivity in blot hybridization is 10 pg for identification of type I viral DNA and 50 pg for type 2 viral DNA. The DNAs of cytomegalovirus and herpes zoster virus do not show positive signals with the probe. The increased sensitivity of the used dot hybridization as compared with biological or IEA antigen identification of the virus was confirmed with the clinical material from 59 patients with the different clinical manifestations of the herpes viral infection.     

Latent herpes simplex virus type 1 DNA contains two copies of the virion DNA joint region.

Southern blot analysis of latent herpes simplex virus DNA detected in mouse brain and digested with a restriction enzyme revealed two copies of the virion DNA joint fragment. Thus, the absence of free ends noted previously in latent herpes simplex virus type 1 DNA is due to joining of the termini.     

Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication

Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.     

Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the L component (UL) of the herpes simplex virus type 1 genome. The second copy of the BamHI-L sequence was inserted in inverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located within UL. A significant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-L sequences at the two different genomic locations. The rearranged genomes either had an inversion of the DNA sequence flanked by the duplication or were recombinant molecules in which different regions of the genome had been duplicated and deleted. Genomic rearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.     

Structure and origin of defective genomes contained in serially passaged herpes simplex virus type 1 (Justin).

Restriction enzyme and hybridization analyses have revealed that high-density DNA prepared from passage 15 of serially passaged herpes simplex virus type 1 (Justin) contains three major classes of modified viral DNA molecules, each composed of distinct but closely related types of repeate units. The DNA sequences within the three types of repeat units are colinear with the DNA sequences located at the right end (between coordinates 0.94 and 1.0) of the parental herpes simplex virus type 1 genome. Thus, the three types of repeat units each contain the entire repeat sequence (ac) (which brackets the unique sequences of the small [S] component of herpes simplex virus type 1 DNA) and differ only with respect to the amount of unique S sequences which they contain. The three classes of high-density DNA molecules were found to be stably propagated between passages 6 and 15 of this series.     

Fine mapping and sequencing of a variable segment in the inverted repeat region of varicella-zoster virus DNA.

A strain variation in the internal and terminal repeats which bind the short unique sequence of varicella-zoster virus (VZV) DNA was found to be due to an insertion or deletion of DNA sequences at a single site. DNA sequence analysis showed that the nucleotide sequence CCGCCGATGGGGAGGGGGCGCGGTACC is tandemly duplicated a variable number of times in different VZV strains and is responsible for the observed variation in mobilities of restriction fragments from this region of VZV DNA. The variable region sequence shares some homology with tandemly repeated regions in the a and c sequences of herpes simplex virus type 1 and probably exists in a noncoding region of the VZV genome.     

Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine.

Mutants of herpes simplex virus type 1 resistant to the antiviral drug 9-beta-D-arabinofuranosyladenine (araA) have been isolated and characterized. AraA-resistant mutants can be isolated readily and appear at an appreciable frequency in low-passage stocks of wild-type virus. Of 13 newly isolated mutants, at least 11 were also resistant to phosphonoacetic acid (PAA). Of four previously described PAA-resistant mutants, two exhibited substantial araA resistance. The araA resistance phenotype of one of these mutants, PAAr5, has been mapped to the HpaI-B fragment of herpes simplex virus DNA by marker transfer, and araA resistance behaved in marker transfer experiments as if it were closely linked to PAA resistance, a recognized marker for the viral DNA polymerase locus. PAAr5 induced viral DNA polymerase activity which was much less susceptible to inhibition by the triphosphate derivative of araA than was wild-type DNA polymerase. These genetic and biochemical data indicate that the herpes simplex virus DNA polymerase gene is a locus which, when mutated, can confer resistance to araA and thus that the herpes simplex virus DNA polymerase is a target for this antiviral drug.     

On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease.

Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases.     

The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.