Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides.

Recombinants between temperature-sensitive mutants of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) were constructed. Using restriction endonucleases, we analyzed the genome composition of 17 intertypic recombinants and detected crossovers in every region of the genome. The virion DNA of one recombinant appeared to be largely "frozen" in two of the four possible genome arrangements of HSV. Knowledge of the genome structures of recombinants enabled us to physically map immediate early polypeptides. We present evidence that the immediate early polypeptide Vmw IE 110 of HSV-1 and its functionally equivalent polypeptide, Vmw IE 118, of HSV-2 may map in the repetitive sequences bounding the long unique region of HSV.     

Quantitation of the viral DNA present in cells transformed by UV-irradiated herpes simplex virus.

Two cell lines biochemically transformed by UV-irradiated herpes simplex virus (HSV) each contain virus DNA. A comparison of the kinetics of reassociation of 3H-labeled HSV DNA in the presence and absence of either clone 139 (HSV-1 transformed) or clone 207 (HSV-2 transformed) DNA showed that the presence of transformed cell DNA increased the rate of reassociation of approximately 10% of the viral genome while having no effect on the remaining 90%. The Cot1/2 of this reaction was approximately 1,000 in each cell type, as compared to approximately 3,000 for the cellular unique sequences. These results suggest the presence of four to six copies of a 10% fragment of the virus DNA per cell. The DNA from a hamster fibroblast cell line morphologically transformed by UV-irradiated HSV-2 (333-8-9) did not affect the rate of reassociation of HSV-2 DNA, indicating that these cells had less than 3% of a viral genome present.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

Detection of viral DNA and RNA by in situ hybridization

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.     

Mouse mammary tumour virus related sequences are present in human DNA

MuMTV-related sequences have been identified in the DNA of human breast cancer cells using the Southern transfer technique and hybridisation with cloned MuMTV DNA under conditions in which partially mismatched sequences form stable hybrids. Hybridisation with cloned fragments of the MuMTV genome showed that the gag-pol region shares the most homology (estimated to be greater than 80%) with the human MuMTV-related sequences, however, DNA fragments partially homologous to the MuMTV LTR, gag ad env regions were also detected. Analysis of several human DNA samples suggests that the majority of the human MuMTV-related sequences are genetically transmitted but additional Eco R1 fragments were detected in the DNA of one out of three breast cancer cell lines, MCF7. These sequences are potential probes for the human MuMTV-related retroviral sequences and will allow their possible role in human breast cancer to be evaluated.     

Physical maps for Herpes simplex virus type 1 DNA for restriction endonucleases Hind III, Hpa-1, and X. bad.

It has been proposed that the genome of herpes simplex virus type 1 (HSV-1) consists of two internal unique sequences, S and L, bounded by two sets of redundant sequences (P. Sheldrick and N. Berthelot, 1974). In this arrangement, terminal sequences (TRs and TRl) are repeated in an internal inverted form (IRs and IRl) and delimit S and L. Furthermore, a body of evidence has accumulated that suggests that S and L themselves are inverted, giving rise to four related forms of the HSV genome. In this study the ordering of restruction endonuclease fragments of HSV-1 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by Hind III, Hpa-1, and X. bad have been constructed for the four related forms of the HSV-1 genome. TRs and IRs were found to be between 3.5 x 10(6) and 4.5 x 10(6) daltons, TRl and IRl about 6 x 10(6) daltons, S about 8 x 10(6) to 9 x 10(6) daltons, and L about 6.8 x 10(6) daltons.     

Cloning of Herpes simplex virus 2 DNA fragments in a plasmid vector

DNA isolated from virions of Herpes simplex type 2 (HSV-2) strain 333 was digested with various restriction enzymes and joined to the EK2 plasmid vector pBR322. The viral DNA sequences present in the hybrids were analyzed by restriction enzyme mapping and hybridization to fragments of HSV-2 DNA. The collection of recombinant molecules represents approx. 75% of the HSV-2-genome. In most cases, the structure of the recombinants seemed identical to the organization of authentic fragments of HSV-2 DNA, however, a few hybrids contained rearrangements of viral and plasmid sequences.     

Isolation of major heat shock protein (hsp70) gene-related sequences from rat genome

Rat genome was assayed for the presence of hsp70 gene-related sequences. Southern blots prepared from rat DNA digested with EcoRI or HindIII restriction endonucleases were hybridized with mouse, human and fruit fly hsp 70 gene probes at increasing stringencies. At the stringency which allows sequences divergent up to about 30% to form stable complexes all three probes detected 25–30 restriction fragments. Increased stringency of the hybridization reduced the number of detectable bands to a few and among them the DNA fragments hybridizing specifically either with mouse or human hsp70 gene probes were detected. Most of the genomic fragments containing hsp70 gene-related sequences were subsequently isolated by screening the rat genomic library with mouse hsp70 gene probe. 168 positive clones were plaque purified and on the basis of the restriction and hybridization pattern we deduced that inserts represented 20 different genomic regions. Partial restriction maps of all isolated genomic fragments were constructed and regions containing hsp70 gene related as well as highly repetitive DNA sequences were localized. A putative sequence rearrangement in the proximity of the hsp70 gene-related sequence was detected in one of the isolated genomic segments.     

The physical state of herpes simplex virus DNA in infected human cells.

Treatment of herpes simplex virus type 1 (HSV-1)-infected human embryo lung (HEL) cells with phosphonoacetic acid (PAA) resulted in complete inhibition of HSV DNA replication. DNA was extracted from PAA-treated HEL cells infected with HSV-1 and centrifuged in a neutral CsCl density gradient. The HSV DNA sequences in the nuclei of PAA treated cells at 24 hr post infection banded at the same density as free HSV DNA (1.725 g/cm3), but a significant amount of viral DNA sequences were detected in the regions of cell DNA (1.700 g/cm3) as well as in the intermediate fractions as determined by hybridization with 3H HSV complementary RNA. The viral DNA sequences of lower deisntiy did not change in density by recentrifugation in a CsCl density gradient, but did change to the density of free viral DNA after treatment with EcoR1 restriction endonuclease. When the DNA from the nuclei of PAA treated cells was analyzed in an alkaline glycerol gradient, more than 95% of the viral DNA sequences were found in the free viral DNA fractions. Since the viral and cellular hybrid DNA represented approximately 33% of the total viral DNA sequences, it is concluded that some of the HSV DNA sequences in PAA treated, infected cells are associated with cell DNA by alkali-labile bonds.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

Identification of the herpes simplex virus DNA sequences present in six herpes simplex virus thymidine kinase-transformed mouse cell lines

We have used a novel filter hybridization approach to detect and map the herpes simplex virus (HSV) DNA sequences which are present in four HSV thymidine kinase (HSVtk+)-transformed cell lines which were derived by exposure of thymidine kinase negative (tk-) mouse cells to UV light-irradiated HSV type 2 (HSV-2). In addition, we have mapped the HSV-1 DNA sequences which are present in two HSV-1tk+-transformed cell lines produced by transfection of tk- mouse cells with sheared HSV-1 DNA. The results of these studies can be summarized as follows. (i) The only HSV DNA sequences which were common to all HSVtk+-transformed cells were those located between map coordinates 0.28 and 0.32. Thus, this region contains all of the viral DNA sequences which are necessary for the expression of HSV-mediated tk transformation. (ii) Many of the cell lines also contained variable amounts of non-tk gene viral DNA sequences located between map coordinates 0.11 to 0.57 and 0.82 to 1.00, suggesting that incorporation of the viral DNA sequences located between these map coordinates is a relatively random event. (iii) The viral DNA sequences located between map coordinates 0 to 0.11 and 0.57 to 0.82 were uniformly absent from all of the HSVtk+ cell lines tested, suggesting that there is a strong negative selective pressure against incorporation of these viral DNA sequences.     

Elimination of specific DNA sequences from the somatic nucleus of the ciliate Tetrahymena

Tetrahymena micronuclear DNA fragments have been cloned in the plasmid pBR322. One clone, pTt 2512, has been found to contain the C-C-C-C-A-A hexanucleotide repeat which is also present in the macronuclear rDNA. Further restriction enzyme digestion and hybridization studies suggest that the clone also contains sequences that are not present in the somatic macronucleus. The flanking sequences of the C4A2 repeats in this clone were separated into four restriction fragments, one from one side and three from the other. These fragments were used as probes for Southern hybridization to study the organizations of similar sequences in the macronucleus and micronucleus. All four fragments hybridized to many fragments of restriction enzyme digested micronuclear DNA. However, none of these hybridizations were detected in the macronucleus. Thus, these families of repetitive DNA are completely eliminated from the macronucleus. Further analysis suggested that the four different sequences may be linked at other locations of the genome. Using nullisomic strains of Tetrahymena, it is found that at least one of these sequences is present in more than one chromosome. Studies of various normal and star strains of Tetrahymena suggest that these sequences are stable in the normal micronucleus but are altered drastically in the defective micronuclei of the star strains. Eliminated DNA of similar nature has also been found in at least five other randomly selected clones of micronuclear DNA and may be present widely in the genome.     

BamI, KpnI, and SalI restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: occurrence of heterogeneities in defined regions of the viral DNA.

We present the locations of the cleavage sites for the BamI, KpnI, and SalI restriction endonucleases within the DNA molecules of herpes simplex virus type 1 (HSV-1) strains Justin and F. These restriction enzymes cleave the HSV-1 DNA at many sites, producing relatively small fragments which should prove useful in future studies of HSV-1 gene structure and function. The mapping data revealed the occurrence of heterogeneity within three regions of the viral genome including (i) the region spanning map coordinates 0.74--0.76, (ii) the ends of the large (L) DNA component, and (iii) the junction between the large (L) and the small (S) components. The heterogeneity in the ends of L and the S-L junctions of HSV-1 (Justin) and HSV-1 (F) DNAs was grossly similar to that previously reported to occur in the ends of L and the S-L junctions of the HSV-1 (KOS) DNA (M. J. Wagner and W. C. Summers, J. Virol. 27:374--387, 1978). Thus, cleavage of these regions with restriction endonucleases yielded sets of minor fragments differing in size by constant increments. However, the various strains of HSV-1 differed with respect to the numbers, size increments, and relative molarities of the various minor fragments, suggesting that the parameters of the heterogeneity are inherited in the structural makeup of the HSV-1 genome. The strain dependence of the pattern of heterogeneity can be most easily explained in terms of variable sizes of the terminally reiterated a sequence, contained in the DNA molecules of these three strains of HSV-1.     

The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences.

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.     

Specific arrangements of human satellite III DNA sequences in human chromosomes

DNA was extracted from various rodent-human somatic cell hybrids that contained single or a few human chromosomes. These DNAs were examined by a combination of restriction endonuclease digestion, gel electrophoresis, and filter hybridisation to radioactive satellite DNA probes following transfer of the denatured restriction fragments from a gel to a nitrocellulose filter. In this way the arrangement of sequences homologous to human satellite III were examined on human chromosomes 1, 7, 11, 15, 22 and X. It was found that the distribution of restriction endonuclease sites within satellite III DNA is different on different chromosomes.     

Cloning and physical mapping of Herpesvirus sylvilagus DNA

The physical map positions for the EcoRI restriction fragments of Herpesvirus sylvilagus DNA were determined using cloned virus DNA fragments as probes for hybridization. Approximately 75% of the virus genome was cloned. The internal location of the repeated nucleotide sequences and the lack of isomeric forms suggested that the virus genome belongs to the class C herpesvirus DNA.     

Mechanism of degradation of duplex DNA by the DNase induced by herpes simplex virus

Reaction intermediates formed during the degradation of linear PM2, T5, and λ DNA by herpes simplex virus (HSV) DNase have been examined by agarose gel electrophoresis. Digestion of T5 DNA by HSV type 2 (HSV-2) DNase in the presence of Mn²⁺ (endonuclease only) gave rise to 6 major and 12 minor fragments. Some of the fragments produced correspond to those observed after cleavage of T5 DNA by the single-strand-specific S1 nuclease, indicating that the HSV DNase rapidly cleaves opposite a nick or gap in a duplex DNA molecule. In contrast, HSV DNase did not produce distinct fragments upon digestion of linear PM2 or λ DNA, which do not contain nicks. In the presence of Mg²⁺, when both endonuclease and exonuclease activities of the HSV DNase occur, most of the same distinct fragments from digestion of T5 DNA were observed. However, these fragments were then further degraded preferentially from the ends, presumably by the action of the exonuclease activity. Unit-length λ DNA, EcoRI restriction fragments of λ DNA, and linear PM2 DNA were also degraded from the ends by HSV DNase in the same manner. Previous studies have suggested that the HSV exonuclease degrades in the 3′ → 5′ direction. If this is correct, and since only 5′-monophosphate nucleosides are produced, then HSV DNase should “activate” DNA for DNA polymerase. However, unlike pancreatic DNase I, neither HSV-1 nor HSV-2 DNase, in the presence of Mg²⁺ or Mn²⁺, activated calf thymus DNA for HSV DNA polymerase. This suggests that HSV DNase degrades both strands of a linear double-stranded DNA molecule from the same end at about the same rate. That is, HSV DNase is apparently capable of degrading DNA strands in the 3′ → 5′ direction as well as in the 5′ → 3′ direction, yielding progressively smaller double-stranded molecules with flush ends. Except with minor differences, HSV-1 and HSV-2 DNases act in a similar manner.