Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA.

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.     

Characteristics of chromatin preparations from herpes simplex virus infected cells

Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, 38/39). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and 38/39 which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl2 were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA co-sedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.     

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.     

Rapid Identification and Typing of Herpes Simplex Virus Types 1 and 2 by a Direct Immunofluorescence Technique

An immunofluorescence (FA) technique has been developed which can identify herpes simplex virus (HSV) in clinical specimens and also type the virus directly as type 1 or type 2. This test, first applied to cervicovaginal specimens obtained from 80 mice genitally inoculated with HSV, indicated a sensitivity approaching 80% in comparison to standard viral isolation methods. A similar sensitivity was found when the test was applied to 185 clinical specimens with adequate cells for staining, which were obtained from a variety of sites of patients with suspect herpetic infection. In only 1 of 6 specimens positive by both FA and culture methods was the HSV type wrongly identified by the FA technique. There were also six specimens which were negative by culture methods but positive by the FA test, indicating a specificity of 91%. It is likely that these are not instances of false-positive tests but of other factors which may have resulted in negative viral isolations by culture methods. As more specific reagents become available, it is anticipated that the FA technique will have wider usage in diagnostic laboratories for the identification and typing of HSV types 1 and 2.     

Molecular epidemiology of DNA viruses: applications of restriction endonuclease cleavage site analysis.

Restriction endonucleases which cleave DNA at specific nucleotide sequences can be used to produce a set of DNA fragments of a viral genome which, when separated by gel electrophoresis, gives a characteristic "fingerprint" for that virus genome. This simple technique has been used to identify and classify DNA viruses of the herpes, adeno, and papova virus groups. Small variants within a given type (e.g., herpes simplex type I) are genetically stable and permit study and identification of individual strains of viruses. Such analyses have recently been applied to study the epidemiology of some DNA virus outbreaks. Restriction endonuclease fingerprinting provides a useful addition to methods for virus identification and classification.     

Localization of a herpes simplex virus neurovirulence gene dissociated from high-titer virus replication in the brain.

Previous studies with the herpes simplex virus type 1 X type 2 intertypic recombinant RS6 suggested that the genomic region from 0.11 to 0.14 map units is involved in neurovirulence (R. T. Javier, R. L. Thompson, and J. G. Stevens, J. Virol. 61:1978-1984, 1987). To study this further, we isolated an RS6-derived herpes simplex virus intertypic recombinant (R13-1) which has a genetic defect within this area. After inoculation into mouse brains, R13-1 was found to be approximately 10,000-fold less neurovirulent than either the wild-type type 1 or type 2 parental virus. However, R13-1 replicated in the mouse brain to titers resembling those of the wild-type parents. Further comparisons with wild-type counterparts indicated that R13-1 expressed equivalent levels of the enzyme thymidine kinase and replicated to intermediate levels in primary mouse embryo fibroblasts maintained at the normal body temperature for mice. Using marker rescue techniques combined with in vivo selection, we found that recombination between unit-length R13-1 DNA and a cloned type 1 DNA fragment spanning the region from 0.11 to 0.14 map units (EcoRI-d, 0.079 to 0.192 map units) generated viruses with a wild-type neurovirulence phenotype. To further refine the genomic region of interest, we performed marker rescue experiments using two EcoRI-d subclones, EcoRI/BamHI dc (0.079 to 0.143 map units) and BamHI/EcoRI and (0.143 to 0.192 map units), representing the left and right halves of the EcoRI d fragment, respectively. In these experiments the EcoRI/BamHI dc clone, but not the BamHI/EcoRI ad clone, yielded recombinant viruses exhibiting wild-type neurovirulence. These results show that at least one herpes simplex virus gene function associated with neurovirulence is located within a 9.1-kilobase region at 0.079 to 0.143 map units of the viral genome. Perhaps more significantly, the results indicate that this neurovirulence property functions independently of high-titer virus replication in the brain.     

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

斑点杂交生物素法检测流行性出血热病毒RNA

为寻找一种用于检测流行性出血热病毒的分子杂交方法,以生物素-7-dATP标记流行性出血热病毒(EHFV)R_(22)株M片段的cDNAR_3克隆作探针,与人源性的EHFVH-114、H-435株RNA基因组进行斑点杂交,得到阳性结果,可检出5pg的cDNA或RNA。此探针与疱疹病毒DNA不出现杂交信号。以上结果说明这种标记探针具有EHFV特异性,可以扩大应用范围,结果还表明动物源性和人源性EHFV均具有共同的保守核苷酸序列。     

Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus type 1 Angelotti DNA

It has been shown earlier that the reiterated regions TRS and IRS bracketing the Us segment of herpes simplex virus type 1 Angelotti DNA are heterogeneous in size by stepwise insertion of one to six copies of a 550-base-pair nucleotide sequence. Considerably higher amplification of this sequence was observed in defective viral DNA: up to 14 copies were detected to be inserted in the repeat units of a major class of defective herpes simplex virus type 1 Angelotti DNA, dDNA1, which originated from noncontiguous sites located in UL and the inverted repeats of the S component of the parental genome. Physical maps were established for the cleavage sites of KpnI, PstI, XhoI, and BamHI restriction endonucleases on the repeats of dDNA1. The map position of the insertion sequence was determined. It was demonstrated that the amplified inserts were not distributed at random among or within the repeats. A given total population of dDNA1 molecules consisted of different homopolymers, each of which contained a constant number of inserts in all of its repeats. Assuming that a rolling-circle mechanism is involved in the generation of full-length defective herpes simplex virus type 1 Angelotti DNA from single repeat units, these data suggest that the 550-base-pair sequence is amplified in the repeats before the replication process.     

Genetic analysis of temperature-sensitive mutants which define the gene for the major herpes simplex virus type 1 DNA-binding protein.

We have assigned eight temperature-sensitive mutants of herpes simplex virus type 1 to complementation group 1-1. Members of this group fail to complement mutants in herpes simplex virus type 2 complementation group 2-2. The mutation of one member of group 1-1, tsHA1 of strain mP, has been shown to map in or near the sequence which encodes the major herpes simplex virus type 1 DNA-binding protein (Conley et al., J. Virol. 37:191-206, 1981). The mutations of five other members of group 1-1 map in or near the sequence in which the tsHA1 mutation maps, a sequence which lies near the center of UL between the genes for the viral DNA polymerase and viral glycoprotein gAgB. These mutants can be divided into two groups; the mutations of one group map between coordinates 0.385 and 0.398, and the mutations of the other group map between coordinates 0.398 and 0.413. At the nonpermissive temperature mutants in group 1-1 are viral DNA negative, and mutant-infected cells fail to react with monoclonal antibody to the 130,000-dalton DNA-binding protein. Taken together, these data indicate that mutants in complementation groups 1-1 and 2-2 define the gene for the major herpes simplex virus DNA-binding protein, an early gene product required for viral DNA synthesis.     

Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system.

After corneal inoculation, herpes simplex virus type 1 replicates in the mouse eye, trigeminal ganglia, and brainstem, producing first an acute and then a latent infection. Previous work from this laboratory focused on the structure of the viral DNA in this system. We have now examined the structure of the viral genome at the chromosome level by using micrococcal nuclease digestion. Studies with disaggregated cell preparations made from the brainstems of acutely infected mice show that the majority of the viral DNA is in a nonnucleosomal form; however, a nucleosomelike fraction was also consistently detected. A similar result was obtained for viral DNA in herpes simplex virus type 1-infected C1300 (clone NA) neuroblastoma cells (a neuronal cell line).     

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.     

Transcription from the BamHI J fragment of herpes simplex virus type 1 (KOS)

RNA transfer experiments (Northern analyses) were used to localize polyadenylated mRNA species made after herpes simplex virus type 1 infection to EcoRI and BamHI fragments and subfragments from the short unique region of the herpes simplex virus type 1 (KOS) genome. Three predominant early mRNAs of 2.5, 1.3, and 0.9 kilobases map in the BamHI J fragment. A detailed restriction map of the BamHI J fragment was constructed.     

Detection of herpes simplex virus type-2 DNA restriction fragments in human cervical carcinoma tissue.

DNA extracted from eight human cervical carcinomas, one lymph node metastasis and related control tissue was examined for the presence of herpes simplex virus (HSV) DNA sequences. Southern blot transfers of tumour and control DNA were hybridised with radioactively labelled cloned probes representing 70% of the HSV-2 genome. Specific hybridisation to HSV DNA sequences was observed in one of eight carcinoma tissues analysed. Hybridisation of HSV-2 DNA probes to BamHI and XhoI restriction enzyme fragments of tumour cell DNA which co-migrated with authentic HSV-2 viral fragments identified co-linear HSV-2 DNA sequences comprising 3% of the HSV-2 genome, between map coordinates 0.582 and 0.612. The remaining eight tumour and all control tissues analysed, showed no specific hybridisation to any of the probes used at levels of sensitivity which would detect 0.5 copies/cell of HSV-2 DNA restriction fragments of 2 kb or greater.     

Herpes simplex virus type 1 Angelotti and a defective viral genotype: analysis of genome structures and genetic relatedness by DNA-DNA reassociation kinetics.

DNA-DNA reassociation kinetics of herpes simplex virus type 1 Angelotti DNA and a class of defective viral DNA revealed that the viral standard genome has a total sequence complexity of about 93 X 10(6) daltons and that a portion of 11 X 10(6) daltons occurs twice on the viral genome. These results agree with structural features of herpes simplex virus type 1 DNA derived from electron microscopic studies and restriction enzyme analyses by several investigators. The defective viral DNA (molecular weight, about 97 X 10(6)) displays a sequence complexity of about 11 X 10(6) daltons, suggesting that the molecule is built up by repetitions of standard DNA sequences comprising about 15,000 base pairs. A 2 X 10(6)-dalton portion of these sequences maps in the redundant region and a 9 X 10(6)-dalton portion maps in the unique part of the standard herpes simplex virus type 1 Angelotti DNA, as could be shown by reassociation of viral standard DNA in the presence of defective DNA and vice versa. No cellular DNA sequences could be detected in defective DNA. A 12% molar fraction of the defective DNA consists of highly repetitive sequences of about 350 to 500 base pairs in length.     

Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1.

A DNA- temperature-sensitive mutant of herpes simplex virus type 1 exhibiting thermolabile DNA polymerase activity, tsD9, was shown to be resistant to phosphonoacetic acid (PAA) when plated at the permissive temperature. ts+ revertants of tsD9 were PAA sensitive and exhibited DNA polymerase activity intermediate between that of the wild-type virus and tsD9, indicating that both temperature sensitivity and sensitivity to PAA are controlled by the same gene. Since the position of tsD9 on the existing herpes simplex virus type 1 linkage map is known, the locus for PAA resistance--and therefore for the structural gene for viral DNA polymerase--has been identified.