Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.     

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.     

Sequence requirements for DNA rearrangements induced by the terminal repeat of herpes simplex virus type 1 KOS DNA.

We investigated the sequence requirements for the site-specific DNA cleavages and recombinational genome isomerization events driven by the terminal repeat or a sequence of herpes simplex virus type 1 KOS DNA by inserting a series of mutated a sequences into the thymidine kinase locus in the intact viral genome. Our results indicate that sequences located at both extremities of the a sequence contribute to these events. Deletions entering from the Ub side of the a sequence progressively reduced the frequency of DNA rearrangements, and further deletion of the internal DR2 repeat array had an additional inhibitory effect. This deletion series allowed us to map the pac1 site-specific DNA cleavage signal specifying the S-terminal cleavage to a sequence that is conserved among herpesvirus genomes. Constructs lacking this signal were unable to directly specify the S-terminal cleavage event but retained a reduced ability to give rise to S termini following recombination with intact a sequences. Deletions entering from the Uc side demonstrated that the copy of direct repeat 1 located adjacent to the Uc region plays an important role in the DNA rearrangements induced by the a sequence: mutants lacking this sequence displayed a reduced frequency of novel terminal and recombinational inversion fragments, and further deletions of the Uc region had a relatively minor additional effect. By using a construct in which site-specific cleavage was directed to heterologous DNA sequences, we found that the recombination events leading to genome segment inversion did not occur at the sites of DNA cleavage used by the cleavage-packaging machinery. This observation, coupled with the finding that completely nonoverlapping portions of the a sequence retained detectable recombinational activity, suggests that inter-a recombination does not occur by cleavage-ligation at a single specific site in herpes simplex virus type 1 strain KOS. The mutational sensitivity of the extremities of the a sequence leads us to hypothesize that the site-specific DNA breaks induced by the cleavage-packaging system stimulate the initiation of recombination.     

Herpes simplex virus cloned DNA fragments induce coumermycin A1 resistance in Escherichia coli.

We have taken a new approach to identify and fine map previously undescribed herpes simplex virus (HSV) functions. In experiments described in this report the antibiotic coumermycin A1 was used to select two HSV type 1 BamHI fragments cloned in pBR322 that confer partial resistance to drug-susceptible Escherichia coli. The genes encoding these HSV functions have been designated cour-1 and cour-2 and have been fine mapped to the HSV sequences. HSV-cour1 is located at the left end of BamHI-F near HSV type 1 genomic map coordinate 0.645. cour-2 maps to BamHI-M', which is a 159-base-pair internal component of the alpha ICP4-coding sequence located in the reiterated sequences of the S component. In pBR322 both inserts apparently rely on the tet promoter for expression. Additionally, cour-2 functions when present as a BamHI insert in pUC7. The analysis of cour-2 "maxi" cell proteins reveals the presence of proteins produced by the fusion of HSV-1 BamHI-M' sequences and the sequences of the vector genes, i.e., the major tet product for pBR322 and the modified beta-galactosidase for pUC7. These data suggest that the development of bacterial assays for fusion products of eucaryotic DNA open reading frames in plasmid vectors may be a useful technique for initiating gene function studies.     

Herpes simplex virus type 1 and 2 (HSV-1,2) DNA detection by PCR during genital herpes

The oligonucleotide primers for herpes simplex virus type 1 and 2 DNA detection are developed. In examined group of patients with genital herpes the virus of type 2 and type 1 was detected in 63% and 26% cases, respectively. The mixed infection of both types is revealed in 11% of the patients.     

Herpes simplex virus type 1 DNA polymerase. Mechanism-based affinity chromatography

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.     

Herpes simplex virus type 1 DNA polymerase. Mechanism of inhibition by acyclovir triphosphate

Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the next deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the "next nucleotide") from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the next nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the next required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the next required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.     

Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were contransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (α0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.     

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.     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.     

Virion component of herpes simplex virus type 1 KOS interferes with early shutoff of host protein synthesis induced by herpes simplex virus type 2 186.

Herpes simplex virus (HSV) strains HSV type 1 (HSV-1) KOS and HSV-2 186 are representative of delayed and early shutoff strains, respectively, with regard to their ability to inhibit protein synthesis in Friend erythroleukemia cells. When these cells were simultaneously infected with HSV-1 KOS and HSV-2 186, HSV-1 KOS interfered with the rapid suppression of globin synthesis induced by HSV-2 186. The observed interference was competitive and not due to exclusion of HSV-2 by HSV-1 at the level of adsorption. Furthermore, UV-irradiated HSV-1 KOS was also effective at interfering with the early shutoff function of HSV-2 186, indicating that a virion component is responsible for the observed interference.     

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.     

DNA sequence of the Herpes simplex virus type 2 glycoprotein D gene

We describe a 1635-bp Herpes simplex virus type 2 (HSV-2) DNA sequence containing the entire coding region of glycoprotein D (gD-2). The amino acid sequence of gD-2, deduced from the nucleotide sequence, was compared to that of the analogous Herpes simplex virus type 1 (HSV-1) glycoprotein (gD-1). The two glycoproteins are 85% homologous and contain highly conserved regions of as much as 49 amino acids in length. Comparison of DNA sequences upstream from gD-1 and gD-2 coding regions identified possible conserved regulatory sequences.     

Herpes simplex virus type 2 US3 blocks apoptosis induced by sorbitol treatment

Previously, we established HEp2 cell lines which express the US3 protein kinase of herpes simplex virus type 2 upon induction with IPTG. Using these cells, we examined whether expression of US3 is sufficient to protect cells from apoptotic cell death induced by sorbitol. Cells expressing US3 showed significantly reduced nuclear fragmentation in the degree that DNA fragmentation and caspase-3 activation were suppressed. It is known that stressors such as osmotic shock and UV irradiation induce the activation of the JNK (c-Jun N-terminal kinase), which can lead to apoptotic cell death. Expression of US3 resulted in the suppression of sorbitol-induced phosphorylation of JNK and MKK4/SEK1, suggesting that the suppression of apoptotic cell death was due to the attenuation of JNK activity.     

Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS.

It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness.     

Herpes simplex virus type 1 ICP27-dependent activation of NF-kappaB

The ability of herpes simplex virus type 1 (HSV-1) to activate NF-kappaB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-kappaB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IkappaBalpha and IkappaBbeta. In this study, we identified loss of IkappaBalpha as a marker of NF-kappaB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IkappaBalpha loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IkappaBalpha loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-kappaB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-kappaB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IkappaBalpha and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-kappaB activation during HSV infection, highlighting the important role that NF-kappaB may play in infection.     

Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences.

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.     

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.