Herpes Simplex Virus Genome Research Articles

Differentiation between alpha promoter and regulator regions of herpes simplex virus 1: the functional domains and sequence of a movable alpha regulator.

The herpes simplex virus genome consists of at least three groups of genes--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. We have established that the elements involved in regulation of alpha genes are a sequence that promotes gene expression and a sequence that confers alpha regulation on the gene by responding to trans-acting regulatory signals. The domains of these sequences were mapped by determining the regulation of thymidine kinase (TK) in L cells converted to TK+ phenotype by chimeric TK indicator genes. The chimeric genes were constructed from appropriate portions of the TK gene fused to donor sequences derived from the 5' nontranscribed and nontranslated leader portions of the viral alpha gene 4. The results were as follows. (i) The natural beta TK indicator extending 5' up to -80 and the chimeric alpha TK extending 5' up to -110 both converted cells to TK+ phenotype but were not regulated. (ii) A segment of the regulator region of the alpha gene 4, extending 5' from position -110, confers inducible alpha-type regulation when fused to the nonregulated but expressible beta TK indicator described above. (iii) The extent of gene induction appears to hinge on the size of the regulatory region inserted into the chimeric gene and correlates with the presence of repeated consensus sequences and G+C-rich inverted repeats in the regulatory region of the alpha gene 4 and other alpha genes.

Limited transcription of the herpes simplex virus genome when latent in human sensory ganglia.

Human paravertebral ganglia were examined for the presence of specific sequences of viral RNA by in situ cytological hybridization, using as probes individual fragments of herpes simplex virus type 2 DNA. Ganglia were obtained at autopsy from 40 patients, and 14 were positive for herpes simplex virus RNA. Transcripts from the left-hand 30% of the viral genome were detected in all of the herpes simplex virus-positive cases. RNA homologous to other sequences from the L component were present less frequently, and no RNA from the S component was detected. These results argue that specific transcription of the herpes simplex virus genome occurs in latently infected human ganglion cells.

Site-specific inversion sequence of the herpes simplex virus genome: domain and structural features.

The genome of herpes simplex virus-1 consists of two covalently linked components, L and S, that invert relative to each other. The L and S components consist of unique DNA sequences bracketed by inverted repeats. The inverted repeats of the L component are designated ab and b' a' and those of the S component are designated a' c' and ca. The number of a sequences at the termini and at the L-S component junction varies from one to several copies. Insertion into the middle of the L component of a DNA fragment consisting of 156 base pairs (bp) of the b sequence, an entire a sequence of 501 bp, and 618 bp of the c sequence created a new site through which additional inversions in the genome occurred. Comparison of the nucleotide sequences of DNA fragments containing one and two a sequences defined the domain of the a sequence. The single a sequence consists of two 20-bp direct repeats (designated as DR1) bracketing a region that contains 19 tandem direct repeats of a 12-bp sequence (DR2) adjacent to three direct repeats of a 37-bp sequence (DR4), in addition to short stretches of unique sequences. The fragment with two tandem a sequences contained three copies of DR1-i.e., the intervening DR1 was shared by the two a sequences. Furthermore, one a sequence contained 22 copies of DR2 and two copies of DR4 whereas the second a sequence contained 19 copies of DR2 and two copies of DR4. These observations suggest that (i) amplification of the number of terminal and internal a sequences is the consequence of intramolecular or intermolecular recombination through DR1, (ii) the number of copies of DR2 and DR4 within the a sequence is not fixed and may vary as a consequence of unequal crossing over or slippage, and (iii) inversion results from intramolecular recombination between terminal and inverted a sequences.

Human cytomegalovirus DNA: Restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs

Complete physical maps for human cytomegalovirus (Davis strain) DNA were constructed from fragments produced by restriction endonucleases HindIII, XbaI, and EcoRI. The results showed that the human cytomegalovirus genome has a structural organization similar to that of the herpes simplex virus genome: a long (L) segment, comprising 82% of the genome, joined to a short (S) segment, comprising the remaining 18% of the genome. Permutations of the S and L segments produce four different molecular arrangements which are represented in equimolar proportions in virion DNA preparations. Heterogeneity in the size of the DNA was detected within fragments which map at one terminus of the S segment and also in fragments which map at one terminus of the L segment. The regions of the genome from which RNA synthesized at various times after infection originates was determined. Immediate-early RNA (RNA synthesized in the presence of cycloheximide) was restricted to a few regions on the genome but hybridized in abundance to a region which mapped between 0.686 and 0.733 units (within the L segment). Early RNA (synthesized in the absence of viral DNA synthesis) hybridized to most areas of the genome, but in particular abundance to a small region which mapped between 0 and 0.046 units (also within the L segment). RNA which accumulated during the late phase of infection was not characterized by particular abundances to any region of the genome.

Isolation of virus from brain after immunosuppression of mice with latent herpes simplex.

Herpes simplex virus (HSV) is usually present in a latent form in the trigeminal ganglion of man1–3. Various stress factors may induce virus reactivation, which is manifest by a lip lesion (innervated from the trigeminal ganglion) and the production of infectious virus. The considerable experimental efforts to define the conditions that lead to the reactivation of latent HSV have concentrated on isolating virus either from the original extraneural site of virus inoculation, or from cell-free homogenates of sensory ganglia from latently infected animals4–15. Recent DNA hybridization experiments resulted in the demonstration of the presence of HSV genomes in the brain tissue of both latently infected mice, and of humans who showed no clinical symptoms of HSV (ref. 16 and N. Fraser, personal communication). This led us to consider the possibility that HSV may be present in brain tissue as the result of either reactivation of the virus in brain cells or the passage of reactivated virus from trigeminal ganglia through the brain stem to the brain. The presence of infectious HSV in brain tissue has not previously been demonstrated; yet this could be a factor in chronic, relapsing neurological diseases such as multiple sclerosis. We have now shown experimentally that mice carrying latent HSV in their trigeminal ganglia may, following massive immunosuppression, express infectious virus in the central nervous system (CNS).

Method for induction of mutations in physically defined regions of the herpes simplex virus genome.

A procedure was developed for inducing mutations in isolated restriction enzyme fragments of herpes simplex virus type 1 (HSV-1) DNA with nitrous acid. The mutations were then transferred to the viral genome by genetic recombination during cotransfection of rabbit kidney cells with the mutagenized fragments and intact HSV-1 DNA. The HpaI restriction enzyme fragments LD, B, LG, I, and J were mutagenized. Temperature-sensitive mutants were found at frequencies of 1 to 5% among the progeny of the transfections. Syncytial mutants also were found at high frequency when fragment B or LD was used for mutagenesis. Fifteen of these mutants, 11 temperature sensitive and 4 syncytial, were used for further studies, including complementation analysis, DNA synthesis, and marker rescue. Marker rescue data presented here and in the accompanying publication (A. L. Goldin, R. M. Sandri-Goldin, M. Levine, and J. C. Glorioso, J. Virol. 38: 50-58, 1981) confirm the map position of some of the newly isolated mutants.

Molecular engineering of the herpes simplex virus genome: Insertion of a second L-S junction into the genome causes additional genome inversions

We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombination between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

Genetic retrieval of viral genome sequences from herpes simplex virus transformed cells.

Oncogenic transformation of cultured cells by inactivated herpes simplex virus (HSV) types 1 and 2 has been demonstrated. Expression of HSV information in these transformed cells has been shown by immunofluorescence studies, detection of HSV neutralizing antibody in sera from tumour-bearing animals and by hybridization of HSV-specific RNA. Molecular hybridization studies of DNA from HSV-2 transformed hamster cells have detected up to 40% of the HSV genome present in several copies. Complementation of three HSV-2 temperature-sensitive mutants when superinfecting the RE1 rat embryo cell line (transformed by the HSV-2 temperature-sensitive mutant ts1) suggests that resident viral genes can be expressed. Brown et al. used a similar approach to detect HSV information latent in human ganglia. We report here retrieval of intertypic HSV recombinants from HSV transformed cells after superinfection with ts mutants of the alternative serotype of HSV. Restriction enzyme analysis which clearly differentiates between HSV-1 and HSV-2 DNA has demonstrated the isolation of recombinants spanning the genome and of virus indistinguishable from the original transforming virus.

Activation of mouse retrovirus by herpes simplex virus type 1 cloned DNA fragments

Cloned DNA fragments from herpes simplex virus (HSV) type 1 (strain Patton) were tested for activation of endogenous mouse retrovirus in BALB/3T3 cells. Activation within the L region of HSV-1 DNA was observed with the ∼3.4-kilobase pair (kbp) BamHI fragment which contains the virus thymidine kinase (TK) gene, and the ∼5.3-kbp EcoRI L fragment. Activation by the TK-containing BamHI fragment was abrogated by digestion with EcoRI. Activation within the S region of HSV-1 DNA was observed with the ∼15.2-kbp EcoRI H ragment and the ∼8.4-kbp Eco RI/ HindIII H/G fragment. Assaying for retrovirus activation serves as an additional parameter for mapping biological functions within the HSV genome.

Distribution of sequences homologous to the DNA of herpes simplex virus, types 1 and 2, in the genome of pseudorabies virus.

The distribution of related sequences between the genomes of pseudorabies virus (PRV) and herpes simplex virus, types 1 (HSV-1) and 2 (HSV-2), was determined. Approximately 7% of the sequences in PRV are shared by HSV-1 and HSV-2 DNAs. By means of the Southern blot technique, it was found that the homologous sequences are not sequestered in one region but are distributed throughout the PRV genome. HSV-1 and HSV-2 have the greatest homology with the long unique region of PRV DNA and the least with the inverted repeat regions of the molecule. HSV-1 DNA also has few sequences homologous to the short unique region of the PRV genome; HSV-2 DNA hybridizes well to this region. There was no homology of HSV-1 or HSV-2 DNAs with the extreme ends of the inverted repeat regions of PRV DNA.

The structure and isomerization of herpes simplex virus genomes

The structure and isomerization of herpes simplex virus genomes

Electron microscopic mapping of proteins bound to herpes simplex virus DNA

Exonuclease digestion experiments have suggested that there is a protein(s) bound close to one or both ends of herpes simplex virus-1 (HSV) DNA. The existence of such bound proteins has been positively demonstrated and their positions on the HSV genome determined by application of a newly developed method for electron microscopic mapping of proteins bound to nucleic acids. Purified HSV DNA was treated with dinitrofluorobenzene under conditions that covalently attach the dinitrophenyl (DNP) group to the proteins in a protein-nucleic acid complex. The HSV DNA-protein-(DNP)n complex was treated with rabbit anti-DNP IgG, and, in some cases, additionally treated with monovalent Fab fragments of goat anti-rabbit IgG, and mounted for examination in the electron microscope. Electron opaque dots representing the protein-(DNP)n-(IgG)m complex were seen on the HSV DNA. Direct measurements of the positions of the protein, as well as partial denaturation mapping, indicate that there are four positions for protein bound to HSV DNA: two near but not at the two ends and two at sites corresponding to the internal inverted repeats of the ends. These results suggest that there is a specific protein binding sequence within the direct terminal repeat of HSV DNA. The previous observation that HSV DNA is more sensitive to digestion by a 3' than by a 5' exonuclease then indicates that the bound protein(s) is more intimately associated with one strand of the specific sequence than with the complementary strand.

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 mapping of herpes simplex virus-induced polypeptides

Analysis of the polypeptides induced by 29 herpes simplex virus type 1/type 2 intertypic recombinants and correlation of the data with the crossover points in the recombinant DNAs have enabled the map positions of many polypeptides to be deduced. These include 25 polypeptides which label with [35S]methionine, 11 which label with [32P]orthophosphate, and 4 which label with [14C]glucosamine. Together with the data of Preston et al. (J. Virol., in press) on the mapping of five immediate-early polypeptides, the results show that representatives of four groups of proteins--immediate-early, late, phosphorylated, and glycosylated--map in both long and short regions. The functional organization of the herpes simplex virus genome does not therefore restrict any of these four groups to either the long or the short region.

Structural studies and physical maps for the herpes simplex virus genome

Structural studies and physical maps for the herpes simplex virus genome

Cross-reacting herpes simplex virus antigens in hamster and mouse cells transformed by ultraviolet light-inactivated herpes simplex virus type 2

Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV -2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provided immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone EC1 3 provided a 53% immunity against itself at an inoculum of 10(6); the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.

Restricted transcription of the herpes simplex virus genome occurring early after infection and in the presence of metabolic inhibitors

There is restricted transcription of the genome of Herpes simplex virus (HSV) prior to viral DNA replication (early after infection). Restricted transcription also takes place in the presence of protein and DNA synthesis inhibitors. We have carried out an extensive kinetic analysis of the rate of solution hybridization between viral DNA and RNA found in cells under these conditions. Results of these analyses have demonstrated that there are two concentration classes of viral RNA synthesized during restricted transcription. The major component is homologous to 20% of the viral DNA; the minor concentration class of viral RNA is homologous to an undetermined portion of the remainder of the viral DNA. Prior to viral DNA replication the second class of RNA comprises no more than 10% of the total concentration of viral RNA. Also, the relative concentration of this second RNA component varies from less than 5% to 15% of total viral RNA synthesized in the presence of DNA and protein-synthesis inhibitors. The concentration of viral RNA early after infection is proportional to the multiplicity of infection. The multiplicity of infection has no effect on the fraction of the HSV genome expressed as major concentration classes of viral RNA. Several models of transcription consistent with these data are discussed.

Transcription of the herpes simplex virus genome in human cells.

We have examined the details of the transcription of the herpes simplex virus (HSV) genome in HeLa cells using deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization. The following findings are reported. (i) Virus-specific RNA (vRNA) synthesized following onset of HSV-DNA replication (L-vRNA) is complementary to as much as 90% of the HSV genome. (ii) There is no significant class of virus-specific RNA synthesized later than L-vRNA. (iii) The vRNA synthesized prior to HSV-DNA replication (E-vRNA) is composed of two classes; one class comprising 75% of the total E-vRNA is found in large amounts as early as 45 min after infection, whereas the other class making up the other 25% of E-vRNA is found in only small amounts at 1.5 hr after infection. This second class of E-vRNA is found in amounts comparable to the first by 3.5 hr after infection. (iv) Inhibition of HSV-DNA synthesis results in the continued synthesis of E-vRNA, but there is no synthesis of L-vRNA. (v) Finally, there is no class of vRNA found in the nucleus that is not found associated with cytoplasmic polyribosomes either early or late after infection.

Evidence for transcriptional control of the herpes simplex virus genome in infected human cells

Evidence for transcriptional control of the herpes simplex virus genome in infected human cells

Herpes vimplex virus: genome size and redundancy studied by renaturation kinetics.

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 +/- 1) x 10(6) daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 +/- 5) x 10(6) daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 x 10(6) to 99 x 10(6) daltons in molecular weight.