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.     

The genome nucleotide sequence of herpes simplex virus 1 strain L2

The genome nucleotide sequence of the reference strain of herpes simplex virus type 1 was obtained using the technique of full size sequencing. For the virus genome structure determination, 402444 reads with an average length of 202 bp were performed, which corresponded to the 542-fold genome coverage. The data were collected to 52 contigs with N50-4518 and the total contig length of 120929 bp. The sequence obtained was deposited into the GenBank database.     

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.     

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.     

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 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.     

Characterization of strain HSZP of herpes simplex virus type 1 (HSV1)

The genetic background of HSZP virus, an HSV1 strain with extensive passage history, was analyzed by parallel comparative sequencing of four relevant genes (UL27/gB, UL41/vhs, UL44/gC and UL53/gK) of HSZP and additional three selected viruses [strains ANGpath, strains KOS(a) and KOS(b) and the prototype strain 17]. Mutation at position 858 (His for Arg) in gB of HSZP was found to be responsible for giant cell formation (syn ³gB mutation) similarly as the 855 mutation (Val for Ala) in the gB of ANGpath. Nosyn ¹gK mutations were detected in the UL53 gene either of HSZP or of ANGpath viruses. The reduced virulence of HSZP for adult mice after peripheral inoculation, similarly as that of KOS virus, seems to be related (at least in part) to numerous mutations in the gB ectodomain. Of these, two mutations located in the antigenic domain IV were the same in gB^HSZP as well as in gB^KOS (at amino acids 59 and 79), at least two (amino acids 313 and 553) were specific for gB^KOS, while one mutation (Ser for Ala at position 108) was specific for gB^HSZP. The abolished shutoff function of the HSZP virus was related to at least four out of six specific mutations seen in thevhs polypeptide (vhs ^HSZP) encoded by the UL41 gene, of which three (amino acids 374, 386, 392) were clustered in the semiconservative box A ofvhs ^HSZP (the truncation of which abrogates the inhibition provided by this protein) and one mutation (at amino acid 18) was situated in the highly conservative locus I ofvhs ^HSZP. In addition, the twovhs ^KOS specific mutations (amino acids 19 and 317) not found invhs ^HSZP, enhanced the early host shutoff function of thevhs ^KOS protein. Finally, gC^HSZP had two specific mutations (amino acids 137 and 147) located in the antigenic domain II of gC, which is responsible for binding of HSV1 virions to the glycoso-aminoglycan (GAG) receptor. When expressed in Sf21 cells using the recombinatt baculovirus system (Bac-to-Bac), gC^HSZP and gC^KOS showed no essential antigenic differences. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. We carried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared our sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

Herpes simplex virus type 1 ANG (HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, mice died of encephalitis within 4 to 5 days after intracerebral inoculation with this strain. HSV-1 ANG was serially passaged in mouse brains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed infections both nonpathogenic and pathogenic viruses replicated at the primary site of infection and spread to various organs. However, only the pathogenic phenotype could be recovered from the spinal cord and the brain. Comparison of the restriction enzyme cleavage patterns of pathogenic ANG and nonpathogenic ANG virus DNAs revealed distinct alterations in the S-segment (US) sequences bounded by coordinates 0.953 and 0.958 in the prototype orientation and by coordinates 0.862 to 0.867 in the IS orientation of the viral genome. However, it is not known whether these alterations are physiologically relevant to the observed changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold excess, the nonpathogenic HSV-1 ANG protected mice against its own pathogenic variant as well as against other pathogenic HSV-1 strains. Pathogenic HSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passages in tissue culture at either high or low multiplicity of infection.     

Genome sequence of Pantoea agglomerans strain IG1

Pantoea agglomerans is a gram-negative bacterium that grows symbiotically with various plants. Here we report the 4.8-Mb genome sequence of P. agglomerans strain IG1. The lipopolysaccharides derived from P. agglomerans IG1 have been shown to be effective in the prevention of various diseases, such as bacterial or viral infection, lifestyle-related diseases. This genome sequence represents a substantial step toward the elucidation of pathways for production of lipopolysaccharides.     

Genome sequence of Mycoplasma hyorhinis strain GDL-1

Mycoplasma hyorhinis impacts swine health and production in many countries, either as a primary pathogen or as a component of a polymicrobial infection. Isolates of this species are also common contaminants of tissue culture lines. The genome sequence of the cell culture isolate M. hyorhinis GDL-1 is presented herein.     

Hand-to-hand transmission of herpes simplex virus type 1

Droplets of tissue culture fluid containing herpes simplex virus type 1 were placed on the palm of the hand. Each 0.01 ml droplet was taken from a stock virus suspension with a titre of 10(7.5) TCID50/0.1 ml. At 0, 15, 30, 60 and 120 min a droplet was firmly touched with the middle finger of the right hand, after which, attempts were made to recover virus from the finger. At 0 min, when the virus-containing droplet was in a liquid state, there was a 100% rate of virus recovery. By 15 min the droplets had dried out, and after touching dried out droplets there was a 40% virus recovery rate, even though experimental procedures demonstrated that infectious virus was present in the dried out droplets at all test times. If the finger was moistened with tap water or saliva, there was a 100% recovery rate of virus after touching dried out droplets, as well as after touching droplets in a liquid state.     

Molecular basis of the glycoprotein-C-negative phenotype of herpes simplex virus type 1 macroplaque strain.

The basis for the inability of the macroplaque (MP) strain of herpes simplex virus type 1 to express mature glycoprotein C (gC) was examined. RNA transfer (Northern) blot analysis with hybridization probes from the region of the herpes simplex virus type 1 DNA known to encode the gC gene indicated that gC mRNA was produced in MP-infected HeLa cells at levels relative to other mRNAs comparable with that seen in KOS-infected cells. Comparative nucleotide sequence analysis of the gC gene from the MP and KOS strains, coupled with the results of recently reported marker rescue experiments, indicates that the inability of MP to produce gC is due to a frameshift mutation in the gC-coding sequence. Because two different (out-of-phase) open reading frames overlap the gC-coding sequence in the region of the mutation, MP mRNA can encode two gC-related polypeptides. Two polypeptides of the predicted size and precipitable by anti-gC antibodies were produced by in vitro translation of MP mRNA. These polypeptides have not been detected in extracts from infected cells with the same antibodies. Comparative nucleotide sequence analyses led to several corrections in the published sequence for the gC gene and the 17,800-molecular-weight polypeptide gene just to the right in KOS DNA. These relatively minor effects on the predicted amino code sequence of gC are tabulated.     

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Key role of template sequence for primer synthesis by the herpes simplex virus 1 helicase-primase

We investigated the effects of ssDNA template sequence on both primer synthesis and NTP hydrolysis by herpes simplex virus 1 helicase-primase. Primer synthesis was found to be profoundly dependent upon template sequence. Although not absolutely required, an important sequence feature for significant production of longer primers (beyond four nucleotides in length) is a deoxyguanylate-pyrimidine-pyrimidine (3'-G-pyr-pyr-5') triplet in the template. The deoxyguanylate serves both to direct primase to initiate synthesis opposite the adjacent pyrimidine and to dramatically increase primer length. While primase synthesized significantly more long primers on those templates containing a G-pyr-pyr triplet, the enzyme still synthesized massive quantities of di- and trinucleotides on many templates containing this sequence. Varying the sequences around the G-pyr-pyr recognition sequence dramatically altered both the rate of primer synthesis and the fraction of primers longer than four nucleotides, indicating that primase must interact with both the G-pyr-pyr and flanking sequences in the template. In contrast to the large effects that varying the template sequence had on primase activity, ssDNA-dependent NTPase activity was essentially unaffected by changes in template sequence, including the presence or absence of the G-pyr-pyr trinucleotide. In addition to hydrolyzing NTPs the NTPase could also hydrolyze the 5'-terminal phosphate from newly synthesized primers.