Herpes Simplex Virus Type 1 Genome Research Articles

Detection of HSV-1 genome in central nervous system of latently infected mice.

Herpes simplex virus type 1 (HSV-1) can establish latent infections in peripheral nerve ganglia and the central nervous system (CNS) of experimentally infected mice. Latent infections of peripheral nervous tissue are characterized by the ability to recover infectious virus from explant cultures of most of the latently infected ganglia. In contrast, infectious virus is infrequently recovered from the CNS of latently infected mice following explant culture, although viral DNA can be detected in CNS tissue. The presence of incomplete or defective viral genomes during the latent CNS infections has been proposed to explain this difference between the two latent infections. To characterize the completeness and the physical state of the latent viral genome, we used Southern blot hybridization to analyse the viral DNA from latently infected mouse ganglia and brains. Our results, reported here, indicate that most, if not all, of the HSV-1 genome is present in latently infected CNS tissue and that the latent viral genome exists in a form other than linear, unit length DNA.

Organization of herpes simplex virus type 1 deoxyribonucleic acid during replication probed in living cells with 4,5',8-trimethylpsoralen.

The structure of herpes simplex virus type 1 (HSV-1) DNA in the nuclei of living infected cells was studied with the DNA photoaffinity probe 4,5',8-trimethylpsoralen. The rate of photobinding to HSV-1 DNA was compared to that of a suitable internal control at different times during infection. The rates of photobinding to DNA packaged in virions, capsids, and prereplicative and postreplicative DNA were characteristically different. By 4 h after infection, after the initiation of DNA replication, the rate of photobinding to HSV-1 DNA increased 4 times relative to the rate of binding to the host DNA. The enhanced rate of photobinding to HSV-1 DNA was maintained at all later times during infection and was not affected when frequent single-strand breaks were introduced in HSV-1 DNA by gamma irradiation of infected cells. The results suggest that the bulk of the replicating herpes DNA is free of torsional tension and that the differing rates of photobinding are attributable to changes in accessibility of the HSV-1 DNA. The results are compatible with previous proposals, based on in vitro studies, that intranuclear HSV-1 DNA is primarily free of nucleosomal organization and suggest that there are few, if any, unrestrained DNA supercoils averaged over the entire HSV-1 genome.

Herpes simplex type 1 DNA in human brain tissue.

Herpes simplex virus type 1 (HSV-1) is known to reside latently in the trigeminal ganglia of man. Reactivation of this virus causes skin lesions and may occasionally infect other tissues, including the brain. To determine whether the brain tissue of humans free of clinical signs of HSV-1 infection contains any trace of HSV-1, we examined the DNA from brain tissue by endonuclease digestion, separation of the fragments by gel electrophoresis, and hybridization with labeled HSV-1 DNA probes. Hybrid bands were detected autoradiographically in experiments using cloned and virion-purified fragments of the HSV-1 genome. HSV-1 DNA sequences were found in 6 of 11 human brain DNA samples tested. In some cases, these bands corresponded to the bands expected for the complete viral genome, whereas others contained bands representing only a part of the genome. In some cases, the terminal fragments could be found, suggesting that the DNA was in a linear, nonintegrated form.

Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.

Characterization of a major late herpes simplex virus type 1 mRNA.

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes.

Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting of head-to-tail reiterations of sequences derived from the end of the S component of the standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to Mr 100 X 10(6) DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA.

Persistence of herpes simplex virus genes in cells of neuronal origin.

The growth characteristics of the KOS strain of herpes simplex virus type 1 (HSV-1) in cell lines of nervous tissues origin were examined in an attempt to develop a tissue culture system mimicking the in vivo state of HSV-1 latency. We have previously reported that the B103 rat brain neuroma cell line is nonpermissive for growth of the KOS strain. In this report, we show that this nonpermissiveness is a temperature- and multiplicity-dependent phenomenon, with minimum virus yields at an elevated temperature and a low multiplicity of infection. Under these conditions, B103 cells survived infection with active wild-type or mutant HSV-1, whereas similarly treated Vero cells were killed. Six independent cultures of B103 cells surviving HSV-1 infection have been established. The surviving cells ceased production of any HSV-1 virus by 14 days postinfection and resumed growth and division at rates comparable to those of uninfected B103 cells. Survivor cells continued to express HSV-1-specific antigens, however, as detected by indirect immunofluorescence and by surface iodination followed by immunoprecipitation and polyacrylamide gel electrophoresis. The survivor cells did not express all of the surface proteins seen on productively infected B103 cells, and they were not susceptible to complement-mediated immune cytolysis with anti-HSV-1 antiserum. These results demonstrate that at least a portion of the HSV-1 genome is being harbored in these survivor cells.

Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus.

The ability to introduce defined mutations at pre-selected sites on DNA molecules in vitro allows one to test hypotheses about the function of specific DNA sequences. The genome of herpes simplex virus type 1 (HSV-1), consisting of 150 kilobase pairs, is probably too large to allow the direct introduction of mutations in vitro. Therefore, to apply these techniques to the study of HSV genes, the feasibility of a two-step procedure was examined. A defined mutation was first introduced into a small segment of HSV-1 DNA which had previously been cloned in the Escherichia coli plasmid pBR322; the mutation was then incorporated into the genome of infectious HSV-1 by DNA-mediated marker rescue. The approach was tested using the viral gene for thymidine kinase (TK)1–3. TK was chosen for several reasons. First, TK is dispensable for viral infectivity in most conditions of cell culture, and efficient selection systems are available for both the TK− and TK+ phenotypes1,3. This facilitates the recovery of TK-deficient mutants and allows rapid and detailed genetic analysis3,4. Second, Stow et al.5 have shown that TK− DNA sequences are readily rescued from DNA fragments into infectious virus. Third, purified HSV-1 TK DNA is able to convert TK-deficient mammalian cells to a TK+ phenotype6, so that the expression of the viral gene can be studied in both the presence and absence of other regulatory viral gene products. Thus, the TK gene provides a favourable system for an examination of the role of specific DNA sequences in the expression of genes in eukaryotic cells. The results presented here demonstrate that an 800-base pair deletion mutation at the TK locus, generated in vitro, was rescued at a high frequency, producing a TK-deficient deletion mutant of HSV-1.

Inhibition of herpes simplex virus type 1 specific translation in cells treated with poly(rI).POLY(RC).

SUMMARY Primary African green monkey kidney cells (GMK) treated with poly(rI).poly(rC) in the presence of DEAE-dextran (‘treated cells’) developed antiviral resistance and concomitantly released interferon into the medium. Treated and untreated cells were infected with herpes simplex virus type 1 (HSV1) in the presence of cytosine arabinoside (araC), and total RNA was isolated and hybridized with purified radio-labelled HSV1 DNA. The intracellular concentration of virus-specific transcripts was not significantly altered in treated cells, but a smaller proportion of the genome of HSV1 hybridized with the extracted RNA. Transcription was similarly restricted when protein synthesis was inhibited by cycloheximide. To analyse virus translation, proteins were radiolabelled between 6 and 10 h after infection and were immunoprecipitated with a pool of human sera and run on SDS-polyacrylamide gels. No virus-specific proteins could be detected in treated cells. In contrast about 25 HSV1-induced proteins were found in infected cells and about 22 proteins in cells infected in the presence of araC. In particular, two virus proteins with apparent mol. wt. of 128000 and 42500 were immunoprecipitated. Since these two were also detected in cells under conditions where elongation of polypeptide chains was non-specifically retarded, it is unlikely that a similar mechanism was responsible for the impaired growth of HSV1 in our treated cells. We conclude that this impairment probably resulted from regulation at the level of virus translation, probably mediated through interferon.

Analysis of viral DNA sequences in hamster cells transformed by herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) DNA was treated with four restriction endonucleases (EcoRI, HincIII, Bgl II, and Xba I) and eight fragments were purified and labeled with 32P in vitro. The kinetics of renaturation of each of the fragments was measured in the presence of DNA extracted from 333-8-9, a hamster cell line transformed by UV light-inactivated HSV-2 strain 333, and from a series of cloned derivatives and their tumor lines. All of the lines examined contained a partial set of viral sequences present at only a few copies per cell. Passage of the cell lines in tissue culture or in animals resulted in partial loss of viral DNA. Two blocks of sequences were present in most of the lines examined; those mapping at positions 21--33 of the HSV-2 genome were detected in seven of seven cell lines tested and those at positions 60--65 were detected in six of eight. Other sequences from the L component can also be present in the DNA of HSV-2-transformed hamster cells.

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.

Isolation and localization of herpes simplex virus type 1 mRNA abundant before viral DNA synthesis

Herpes simplex virus type 1 (HSV-1) DNA covalently bound to cellulose was used as a reagent to isolate viral RNA transcripts for size analysis on denaturing agarose gels. Nuclear and polyribosomal RNA isolated at 2 h postinfection (p.i.) migrated with sizes between 1,500 and 5,500 nucleotides. At 6 h p.i. (when viral DNA synthesis is underway), viral polyribosome-associated polyadenylated RNA showed different discrete sizes of species predominating, with RNA larger than 5,500 nucleotides clearly present. Nearly 50% of the newly made viral RNA found in the nucleus at 6 h p.i. was from 5,000 to 10,000 nucleotides in length. A high-resolution transcription map of the viral mRNA abundant at 2 h p.i. was compiled from the hybridization of Southern blots of HSV-1 DNA restriction fragments to both sizes of fractionated polyribosomal polyadenylated RNA and 3' complementary DNA probe made to this size of fractionated RNA. We have identified and mapped 16 mRNA species abundant at 2 h p.i. These RNAs range in size from 1,500 to 5,300 nucleotides and map throughout the HSV-1 genome. In some instances, a direction of transcription can be suggested. Further, about one-third of this number of mRNA's has been found in cells infected with a DNA-negative temperature-sensitive mutant (tsB2) and grown at the nonpermissive temperature (39 degrees C).

Presence of herpes simplex virus type 2 DNA in hamster cells oncogenically transformed by ultraviolet-inactivated virus

Herpes simplex virus type 2 (HSV-2) DNA has been detected by molecular hybridization in hamster fibroblast cells oncogenically transformed by ultraviolet-irradiated virus. At early passages after cloning in soft agar, about 40% of the HSV-2 genome was present in all the transformed cell lines at one to six copies per cell. In cell lines derived from tumors induced by these cells, the same percentage of the HSV-2 genome was also found with more uniform number of copies (between two and three). Thus the presence of viral DNA seems to be necessary for the maintenance of the transformed state in these cell lines.

Partial characterization of herpes simplex virus type 2 (HSV-2)-specific poly(A)+ RNA by hybridization to EcoI-generated HSV-2 DNA fragments

Polyadenylated RNA appearing in polyribosomes from Herpes simplex virus type 2 (HSV-2)-infected cells has been isolated at different stages of infection. The amounts of virus-specific RNA hybridizing to individual EcoRI fragments of HSV-2 DNA have been detected. Templates coding for “early” virus-specific RNA are widely dispersed within the HSV-2 genome in the short and long segment since each of the EcoRI fragments showed hybridization with “early” isolated poly(A)+ RNA. The virus-specific fraction of labeled poly(A)+ RNA which hybridized to each of the EcoRI fragments increased with 1-β- d-arabinofuranosylcytosine-treated (Ara-C) poly(A)+ RNA as well as with “late” poly(A)+ RNA. Poly(A)+ RNA from infected, Ara-C-treated cells contained virus-specific RNA which hybridized to each of the EcoRI fragments. We found that the poly(A)+ RNA, specific for one EcoRI fragment, varied with the stage of infection, comparing it to other fragment-specific poly(A)+ RNAs.

Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome

Isolated restriction endonuclease fragments of the herpes simplex virus type 1 (HSV-1) genome were introduced into hamster embryo cells to identify DNA sequences capable of transforming the cells with respect to acquisition of properties correlated with tumorigenicity. One of the fragments generated by cleavage of HSV-1 DNA with the restriction endonuclease Xba I was found to induce transformation at a frequency of about 10 colonies per quantity of fragment recovered from 1 μg of uncut DNA; fractions containing the other Xba I fragments failed to induce transformation reproducibly, although occasional colonies were detected. The fragment with transforming activity (Xba I-F) is 15.5 × 10 6 daltons in molecular weight and is located between 0.30 and 0.45 map units on the HSV-1 genome. The Xba I-F transformants obtained were selected for their ability to replicate in low concentrations of serum; in addition, they were found to attain high saturation densities in the presence of 10% serum and to form colonies in semisolid medium. Moreover, the transformed cells produced at least one of the viral gene products (a membrane glycoprotein) encoded in the fragment used for transformation, indicating not only that viral DNA was incorporated into the cells, but also that viral genes were expressed.

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.

Transformation of hamster embryo and human embryo cells by temperature sensitive mutants of herpes simplex virus type 2

Transformation of hamster embryo (HaE) cells and human embryo (HuE) cells was attempted with temperature sensitive mutants of herpes simplex virus type 2 (HSV-2) at the nonpermissive temperature of 38.5°. Among 12 ts mutants tested, transformation of HaE cells has been observed with three mutants ( ts 4, 9, 35), and of HuE cells with one mutant ( ts 9). Inoculation of transformed HaE cells into newborn hamsters resulted in tumor formation. The continued presence of the HSV-2 genome in transformed HaE and HuE cells was suggested by indirect fluorescent antibody staining using anti-HSV rabbit serum and antirabbit goat serum.

Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultraviolet-irradiated herpes simplex virus type 2.

An in vitro method which led to the transformation of hamster embryo fibroblasts after exposure to herpes simplex virus type 2 (HSV-2) inactivated with ultraviolet irradiation is described. The transformed cells (333-8-9) produced tumors when inoculated into newborn Syrian hamsters but not when injected into weanling Syrian hamsters of the same LSH inbred strain. However, after one in vivo passage, the 333-8-9 cells became highly oncogenic in weanling hamsters. No infectious virus was recovered from these cells. Herpes simplex virus antigens were detected in the transformed cells by the indirect immunofluorescence technique. Sera from tumor-bearing hamsters contained antibody with highly specific neutralizing activity against HSV-2. These studies indicate the continued involvement of the HSV-2 genome in an oncogenic cell line.