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.     

Heterogeneity ofBamHi DNA fragments B and E in several HSV-1 strains and recombinants

The restriction cleavage sites of theBamHI-B andBamHI-E DNA fragments of several Herpes simplex virus type 1 (HVS-1) strains were mapped. These fragments are situated at the ends of the long unique regions and share homologous sequences in the repeat components (TR_L and IR_L) of the genome. All the strains analyzed were found to have deletions in the Hpal-P fragment, situated in theBamHI-B fragment. Five strains were further analyzed and the deletions were located in the Smal-A fragment (within the Hpal-P fragment). TheBamHI-E fragment of four recombinants (obtained by recombination between the HFEM genome and theBamHI-B fragment or part of it from the HSV-1 F strain) were almost identical but differed from another strain [NIH(LP)]. Comparison of theBamHI-B and theBamHI-E fragments of the same strain revealed that the fragments were not identical in all cases.     

Herpes simplex virus type 1 restriction fragment polymorphism determined using southern hybridization

Summary Regions of herpes simplex virus type 1 (HSV-1) DNA with variation in the size of restriction endonuclease fragments were identified by comparison of theBam HI,KpnI orSalI restriction endonuclease digestion patterns among 15 HSV-1 isolates after hybridization with specific³²P-labeled cloned HSV-1 DNA fragments. Of the types of restriction fragment polymorphism identified, one was a strain with a distinctly different restriction fragment than the prototype (loss or gain of restriction sites). Another type, the specific fragment varied only in size among strains. Thirteen distinct variations were identified. Ten were mapped to the unique sequence of the L component; two to the inverted repeat of the L component and one to the inverted repeat of the S component. The presence of a common ancestor from which some isolates of HSV-1 might derive was deduced from an analysis of the distribution of the thirteen variations among the 15 HSV-1 isolates.With 8 FiguresOn leave from the Chemo-Sero-Therapeutic Institute, Kumamoto, Japan.     

Importance of theHpaI-P sequence for herpes simplex virus-1 replication in the adrenal glands

Summary The ability of several strains and recombinants of herpes simplex virus 1 (HSV-1) to proliferate in the adrenal glands and to invade the spinal cord was studied. After intraperitoneal infection, pathogenic HSV-1 strains replicated in the adrenal glands, penetrated the spinal cord and migrated to the brain. The nonpathogenic strain HFEM could not replicate in the adrenal glands, but the recombinant virus MLC1 was able to do so after rescue by reinsertion of theHpaI-P sequence into theBamHI fragment of HFEM DNA. However the recombinant MLC1 virus could not penetrate the spinal cord.The effect of HSV-1 infection on the expression of the cellular genes for multidrug resistance (in the adrenal glands) and proenkephalin A (in the spinal cord) was also studied.     

Genomic characterization of molluscum contagiosum virus type 1: Identification of the repetitive DNA sequences in the viral genome

The genomes (188 kbp) of the prototypeMolluscum contagiosum virus type 1 (MCV-1) and a variant strain (MCV-1v) were characterized by construction of the physical maps of the viral DNA for the restriction enzymesBamHI,ClaI,EcoRI, andHindIII using a defined gene library harboring the DNA sequences of the MCV-1 genome and by DNA-DNA hybridizations. It was found that the genomes of both MCV strains are identical, with the exception of very few changes in the DNA fragmentation patterns of restriction endonucleaseBamHI as a consequence of naturally occurring nucleotide exchanges in the genome of the variant strain. Detailed hybridization experiments revealed the existence of repetitive DNA sequences, which are located within the terminal regions of the viral genome at the map coordinates 0 to 0.027 and 0.973 to 1.     

Strategy for identifying the gene encoding the DNA polymerase of molluscum contagiosum virus type 1

Molluscum contagiosum virus (MCV) is a member of the family Poxviridae and pathogenic to humans. MCV causes benign epidermal tumors mainly in children and young adults and is a common pathogen in immunecompromised individuals. The viral DNA polymerase is the essential enzyme involved in the replication of the genome of DNA viruses. The identification and characterization of the gene encoding the DNA polymerase of molluscum contagiosum virus type 1 (MCV-1) was carried out by PCR technology and nucleotide sequence analysis. Computer-aided analysis of known amino acid sequences of DNA polymerases from two members of the poxvirus family revealed a high amino acid sequence homology of about 49.7% as detected between the DNA polymerases of vaccinia virus (genus Orthopoxvirus) and fowlpoxvirus (genus Avipoxvirus). Specific oligonucleotide primers were designed and synthesized according to the distinct conserved regions of amino acid sequences of the DNA polymerases in which the codon usage of the MCV-1 genome was considered. Using this technology a 228 bp DNA fragment was amplified and used as hybridization probe for identifying the corresponding gene of the MCV-1 genome. It was found that the PCR product was able to hybridize to theBamHI MCV-1 DNA fragment G (9.2 kbp, 0.284 to 0.332 map units). The nucleotide sequence of this particular region of the MCV-1 genome (7267 bp) between map coordinates 0.284 and 0.315 was determined. The analysis of the DNA sequences revealed the presence of 22 open reading frames (ORFs-1 to-22). ORF-13 (3012 bp; nucleotide positions 6624 to 3612) codes for a putative protein of a predicted size of 115 kDa (1004 aa) which shows 40.1% identity and 35% similarity to the amino acid sequences of the DNA polymerases of vaccinia, variola, and fowlpoxvirus. In addition significant homologies (30% to 55%) were found between the amino acid sequences of the ORFs 3,-5,-9, and-14 and the amino acid sequences of the E6R, E8R, E10R, and a 7.3 kDa protein of vaccinia and variola virus, respectively. Comparative analysis of the genomic positions of the loci of the detected viral genes including the DNA polymerases of MCV-1, vaccinia, and variola virus revealed a similar gene organization and arrangement.     

Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM.     

Comparative genome mapping of bovine encephalitis herpesvirus,bovine herpesvirus 1, and buffalo herpesvirus

Summary A clone library of 11 of 15BamHI fragments representing 81% of the 140 kilobase DNA genome of the prototype bovine encephalitis herpesvirus strain N569 (BEHV.N569) was constructed. The clones were used to verify theBamHI,BstEII,EcoRI, andHindIII genomic maps for BEHV.N569 published by Engels et al. [Virus Res 6: 57–73 (1986)] for the same virus although some amendments/variations to theBamHI map were found in that 3 previously unidentified restriction sites were identified. Restriction site maps forBglII andKpnI were also derived for BEHV.N569.Southern blot analysis using³²P-labelled BEHV DNA as probe indicated that bovine herpesvirus 1 (BHV1), buffalo herpesvirus 1 (BuHV1) and caprine herpesvirus 1 (CaHV1) were similar and that the similarity occurred throughout the entire length of the genomes; CaHV1 was more distantly related to the other 3 viruses. Because of the similarities BEHV.N569 and BHV1.Cooper cloned DNA fragments were used to constructBamHI,BglII,BstEII,EcoRI,KpnI, andHindIII restriction site maps for the genome of BuHV1 andBamHI,BglII, andKpnI maps for the genome of BHV1.V155, a genital strain.     

The region 0.7615–0.796 m.u. of the HSV-1 genome determines suppression of humoral antibody formation against herpes simplex virus

Summary The influence of genetic properties of parts of the HSV-1 genome on suppression of humoral antibody formation was investigated by using intratypic recombinants. The deleted strain HFEM (HSV-1) induces suppression. The MluI DNA fragment (coordinates 0.7615–0.796 m.u.) derived from the antibody inducing strain F1 (HSV-1) was transfected into the deleted strain HFEM to produce the recombinant virus R-MlCI and shown to restore antibody formation, as demonstrated by neutralization- and ELISA-tests. The intratypic recombinant viruses R-15, R-19 and R-26, produced by transfection of the Bam HI DNA-fragment B (0.738–0.809 m.u.) of strain Fl into the deleted strain HFEM, resulted in antibody formation only in the recombinant virus R-26. The reason for these different properties might be associated with the presence of small deletions in the Sma I A-fragment (0.763–0.765 m.u.) or elsewhere in the Bam HI DNA-fragment B. Our results were finally correlated to replication of the recombinant viruses in macrophages and to spread into spleen and adrenal glands. There is evidence that antibody formation may be correlated to the ability of HSV to replicate in macrophages.     

Diverse mechanisms of plant resistance to cauliflower mosaic virus revealed by leaf skeleton hybridization

Summary Purified virion DNA of an Australian isolate of equine herpesvirus 4 (EHV 4.405/76) was digested with restriction enzymes and the DNA fragments were cloned into pUC 19. The resulting recombinant plasmid library, representing 92% of the virus genome, was used in hybridization analyses to construct restriction maps forBam HI,Eco RI, andSal I for the EHV 4 genome. The results show that the genome of EHV 4.405/76 was approximately 145 kb and comprised a unique long (U_L) region of 112 kb and a unique short (U_S) region of 12.4 kb. U_s is flanked by an internal and terminal repetitive sequence (IR_s and TR_s) of about 10.3 kb. TheBam HI andEco RI restriction maps are similar to those previously published for an English isolate EHV 4.1942 strain [4] although some differences such as location of an additional fragment and changes in positions of two other small fragments were found.     

Mapping of the deletion in the genome of HSV-1 strain HFEM responsible for its avirulent phenotype

To determine the exact map position of the deletion in the genome of HSV-1 strain HFEM, the Bam HI DNA fragment B of the HSV-1 strains HFEM, F, and of HSV-R-HFehx-C19 were cloned in the Bam HI site of the bacterial plasmid vector pAT153 and analysed in detail. Analysis of the insert of the three recombinant plasmids harbouring the Bam HI DNA fragment B of HSV-1 strain F (pHSF-B-B), HSV-1 strain HFEM (pHSHF-B-B), and recombinant HSV-R-HFehx-C19 (pHSR19-B-B) was performed using a variety of restriction enzymes and Southern blot hybridization. These comparative studies revealed that the Hpa I DNA fragment P of the intact viral genome of HSV-1 strain F is not present in the genome of HSV-1 strain HFEM, indicating that the deletion in HSV-1 strain HFEM corresponds to this fragment, which spans the coordinates 0.762 to 0.790 of the HSV-1 DNA. The size of the deletion was found to be 4.1 kbp, corresponding to 2.7 Md.     

Human T-cell leukemia virus type 1 protease protein expressed inEscherichia coli possesses aspartic proteinase activity

Summary We amplified the human T-cell leukemia virus type 1 (HTLV-1) protease gene fragment by polymerase chain reaction (PCR) and cloned it into a pUC plasmid vector. DNA sequencing data of the protease gene fragment indicated that it contained an open reading frame capable of encoding the active HTLV-1 protease. To express a fusion protein of -galactosidase linked with the HTLV-1 protease inEscherichia coli, a plasmid DNA was constructed by inserting the HTLV-1 protease gene DNA into a procaryotic expression vector, pUEX2, consisting of alacZ gene directed by a phage Pr promoter and designated pUEX-pro. By Western blot analysis using anti--galactosidase antibody, a bigger molecular size band than that of the control -galactosidase molecule was observed inE. coli cells transformed with pUEX-pro but not with control pUEX 2, suggesting that the particular fusion protein was successfully expressed. This recombinant protease protein in theE. coli cell lysate was demonstrated to be able to cleave the decapeptide substrates composed of amino acid sequences containing proteolytic cleavage sites in the HTLV-1gag precursor polyprotein. Thegag precursor polyprotein expressed in the mammalian cells by the recombinant vaccinia virus system was also expectedly cleaved by this enzyme. Significant inhibition of this protease activity by pepstatin A, an aspartic proteinase-specific inhibitor, confirms that HTLV-1 protease is a member of the aspartic proteinase group as suggested previously. Since the crude lysate without purification is utilized sufficiently as a native HTLV-1 protease reagent, this protease preparation is easily applicable to the large scale screening of HTLV-1 protease inhibitors for the treatment of diseases caused by HTLV-1.     

Viable variants in VA-RNA, gene of an Ad2-Ad5 recombinant

We have developed a simple method for selecting viral mutants whose DNA lack one of the sites of a restriction endonuclease that has more than one site on the viral genome. We have used this method for isolating viable variants of an Ad2-Ad5 recombinant that lack a BamHI cleavage site located at mp 29 spanning the VA-RNA_I gene. The viral DNA-protein complex was prepared from a stock of the Ad2-Ad5 recombinant passed under high m.o.i., and cleaved with BamHI. We would expect BamHI to cleave the parental Ad2-Ad5 recombinant DNA at two sites, 29 and 59.5, but to cleave naturally arising mutant DNAs at one or the other site. With mutation at mp 29 site, BamHI cleavage would produce a fusion fragment from mp 0–59.5. Our objective, then, was to recombine the mutant DNA fragment in vivo with an overlapping fragment containing sequences from the right half of the genome, e.g., SalA (mp 45.9–100) to produce an infectious mutant DNA with a single BamHI site at mp 59.5. DNA protein complex was digested with BamHI and cotransfected with the DNA-protein complex cleaved with SalI, and the resulting plaques were screened for DNAs lacking the BamHI site at mp 29. Two such mutants were isolated, one of which is probably a deletion mutant (203) and the other an insertion mutant (204). Mutant 203 made VA-RNA_I of slightly higher molecular weight and mutant 204 made VA-RNA_I of slightly higher molecular weight. These mutants grew to levels comparable to that of the parental virus in KB cells indicating that these mutations did not alter the growth of the mutants. The method described here should also be valuable in isolating mutants elsewhere in the genome.     

Identification of a region of the Epstein-Barr virus (B95-8) genome required for transformation

In a previous study the identification of a region(s) of the Epstein-Barr virus (EBV) genome, which is associated with transformation, was attempted by marker rescue. A transforming EBV was rescued from D98/HR-1 hybrid cells, which contain the nontransforming HR-1 EBV genome, after transfection with specific BamHI and Charon 4A fragments (J. Stoerker and R. Glaser, Proc. Nat. Acad Sci. USA 80, 1726–1729, 1983). In this study, characterization of the EBV DNA in four human lymphoblastoid cell lines (LCL) transformed with rescued virus was performed. It was found that recombination between the transfected fragments, BamHI H,F,X and the Charon 4A fragment (EB-2636) which is equivalent to the BamHI H,F,X region, and the endogenous HR-1 EBV genome in the D98/HR-1 cells took place. This recombination resulted in the formation of transforming EBV. The EBV DNA in the four LCLs are similar to each other and to HR-1 EBV DNA. However, the EBV DNA in all four LCLs also contain the U2 region plus additional sequences of B95-8 DNA. The U2 region is deleted in HR-1 EBV DNA which :is associated with HR-1 cells and the D98/HR-1 hybrid cells. Thus, transforming activity of the HR-1-like viruses rescued from D98/HR-1 cells was concomitant with the recombination of the 0.26–0.36 region of the EBV genome, suggesting that this region is necessary for at least the initiation of transformation.     

Homology of the HSV-2 “a-sequence” to cellular sequences

Bgl-II fragments of the genome of Herpes simplex virus type 2 (HSV-2) HG-52 were cloned into the vector p-Neo and were used to screen the complete HSV-2 genome for regions cross-hybridizing with the genome of HEL cells. Most extensive cross-hybridizing activity was observed with a 530 bpSstII subfragment of the viralBamHI G DNA-fragment (contained inBgl II F), which spans the joint and the viral a-sequence. From a -L47 library, a cellular 15 kbHindIII DNA fragment was subcloned in pBR 322 which contained a 1920 bpSstII subfragment having strong cross-hybridizing activity with the 530 bpSst II fragment of HSV-2BamHI G. Within this 1920 bpSst II fragment the cross-hybridizing activity was confined to a 230 bpBgl I/Hpa II subfragment. This 230 bp fragment (including the flanking sequences) was analyzed in comparison to the viral a-sequence. Sequence data revealed a (G+C) content of 66% in the cellular and 81% in the viral DNA fragment, which is mainly determined by an extremely (G+C) rich 16-fold direct repeat (DR2) at the 5-end. The homology between both DNA-fragments varies between 56% and 79% within the L-S inversion region. Both sequences, furthermore, show homology to the human c-myc protooncogene.     

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D

Summary The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirusAutographa california nuclear polyhedrosis virus (AcNPV). After co-transfection ofSpodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.