The minimal transforming fragment of HSV-2 mtrIII can function as a complex promoter element

The minimal transforming fragment (486 TF) of HSV-2 mtrIII (0.567-0.570 map units) is composed of two distinct and non-overlapping promoter elements when linked to bacterial CAT genes. A 230-nucleotide fragment of 486 TF, Sal1-Hpa1, was active as a promoter element in primate cells but not rodent cells. A 173-nucleotide fragment, Sma1-Pst1, was active in both primate and rodent cells. The 486 TF did not compete for limiting cellular factors required to drive the CAT gene under control of the SV40 early promoter/enhancer. However, gel-retardation assays suggest that unique factors exist in cells transformed by HSV-2 which specifically recognized regions of 486 TF. These results are discussed with respect to HSV-2-mediated transformation.     

Genomic location of bovid herpesvirus type 2 nucleotide sequences homologous to five herpes simplex virus type 1 genes

The location of nucleotide sequences within the bovid herpesvirus 1 (BHV-2) genome homologous to herpes simplex virus 1 (HSV-1) DNA were investigated. BHV-2 DNA was digested with restriction endonucleases and blotted to nitrocellulose paper. The blots were then probed with plasmids containing HSV-1 genes for thymidine kinase (TK), the major DNA binding protein (ICP8), the major capsid protein (VP5) and genes for HSV-1 glycoproteins gB, gD, and gC. Except for HSV-1 gC, each HSV-1 gene tested hybridized to BHV-2 nucleotide sequences that were located either on both sides of a restriction endonuclease cleavage site, within a small restriction endonuclease fragment, or to an area common to two overlapping restriction fragments. Thus, we were able to localize BHV-2 nucleotide sequences homologous to the HSV-1 ICP8 gene between 0.38 and 0.41 map units (m.u.), and BHV-2 nucleotide sequences homologous to the HSV-1 VP5 gene between 0.24 and 0.27 m.u. In addition, BHV-2 nucleotide sequences homologous to HSV-1 genes for TK, gB and gD were found to lie on both sides of restriction endonuclease cleavage sites at 0.30 0.35, and 0.94 m.u., respectively.     

Determination of the nucleotide sequence flanking the deletion (0.762 to 0.789 map units) in the genome of an intraperitoneally avirulent HSV-1 strain HFEM

Herpes simplex virus type 1 (HSV-1) strain HFEM which harbours a deletion of 4.1 kbp in its genome (0.762 to 0.789 map units, HpaI DNA fragment P of HSV-1), is apathogenic for mice and tree shrews by the intraperitoneal application route. The exact position of this deletion was determined by DNA sequence analysis. This analysis was performed using the recombinant plasmid pU18HSHF-XmI-B which harbours the flanking genome regions (0.752 to 0.762 and 0.789 to 0.7895 map units) of the deletion in the genome of HSV-1 HFEM, and the recombinant plasmids pU18HSF-XmI-B, pU18HSF-AS, and pHSF-BB-BsH-D, harbouring particular regions of the genome of the virulent HSV-1 strain F at the coordinates 0.752 to 0.761, 0.786 to 0.790, and 0.762 to 0.771, respectively. The comparison of the DNA sequence of this region with the DNA sequences of the corresponding genome regions of the pathogenic HSV-1 strain F and HSV-1 strain 17 showed that the 5' end of the deletion in the genome of HSV-1 HFEM starts at the nucleotide position 3774 of the BamHI DNA fragment B from HSV-1/17. This position is 71 bp upstream of the UL/RL junction of the HSV-1 genome. The 3' terminus of the deletion ends at the nucleotide position 7226 of the BamHI DNA fragment B from HSV-1/17. The position is within the incomplete ninth repetitive box (ACTCC-CACGCACCCCC) and is located 36 bp upstream of the 3' end of the IE 110 mRNA.     

Virus-specific DNA sequences present in cells which carry the herpes simplex virus thymidine kinase gene.

Two independently derived cell lines which carry the herpes simplex type 2 thymidine kinase gene have been examined for the presence of HSV-2-specific DNA sequences. Both cell lines contained 1 to 3 copies per cell of a sequence lying within map co-ordinates 0.2 to 0.4 of the HSV-2 genome. Revertant cells, which contained no detectable thymidine kinase, did not contain this DNA sequence. The failure of EcoR1-restricted HSV-2 DNA to act as a donor of the thymidine kinase gene in transformation experiments suggests that the gene lies close to the EcoR1 restriction site within this sequence at a map position of approx. 0.3. The HSV-2 kinase gene is therefore approximately co-linear with the HSV-1 gene.     

Cloning and fine mapping the DNA of equine herpesvirus type one defective interfering particles

Equine herpesvirus type one (EHV-1) defective interfering (DI) particle DNA fragments were inserted into the XbaI site of the plasmid vector pACYC184. Five DI XbaI fragments, which ranged in molecular weight from 4.5 to 6.7 MDa, were selected for detailed analysis. Each DI DNA clone was labeled with 32P-deoxynucleotides by nick translation and hybridized to genomic digests of EHV-1 standard (STD) DNA bound to nitrocellulose. All five clones were shown to hybridize to DNA sequences derived from the left terminus (0.0-0.04 map units) of the long (L) region and from the short (S) region inverted repeats (IRs, 0.79-0.86 and 0.93-1.00 map units) of the STD EHV-1 genome. Restriction enzyme mapping studies and Southern blot hybridizations employing cloned STD virus DNA fragments as probes revealed that these EHV-1 DI clones contain two major domains: (1) an L terminal region which maps to 0.01-0.04 map units and is highly conserved among all five clones, and (2) a region homologous to the IRs which appears to vary between individual clones.     

Quantification of the herpes simplex virus DNA present in biochemically transformed mouse cells and their revertants.

Four cell lines biochemically transformed by u.v.-irradiated herpes simplex virus contain virus DNA fragments ranging from 3 to 22% of the HSV genome. Of five revertant clones selected for 3H-TdR or BrdUrd resistance, four had lost all detectable virus DNA while the fifth, selected for BrdUrd resistance, retained the entire virus fragment but there was a reduction of virus copies per cell from 5 to 1. Three 'supertransformed' revertant cell lines contained virus DNA fragments ranging from 12 to 28%. The number of virus DNA fragments per cell ranged from 1 to 5 and clearly indicated that a single copy of the virus thymidine kinase gene is adequate for biochemical transformation. The determination of the base composition of the transforming virus DNA fragment indicated that the transforming DNA has a base composition approximately the same as the HSV genome and does not constitute a low GC virus DNA region. Cross hybridization between HSV-1 transformed cells and HSV-2 DNA is very slight, indicating that the DNA found in clone 139 is not entirely composed of the HSV-1 and HSV-2 common sequences.     

Biochemical transformation of LM(TK-) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK^?)cells to the TK⁺ phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 mM thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK^?)/TF pMH110 E2 and LM(TK^?)/TF pMH110 Hc2] with [³⁵S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK^?) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.     

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK^?)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK^?) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA⁺, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK^?) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA⁺ as well as TK⁺. The HANA⁺ cells included LM(TK^?)/TF pAGO PP and LM(TK^?)/TF pAGO PS clones. The latter are clones of LM(TK^?) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 10⁶ daltons) and a PvuII-SmaI fragment (0.9 × 10⁶ daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.     

Herpes simplex virus types 1 and 2: comparison of the defective genomes and virus-specific polypeptides.

Undiluted serial passage of HSV-1 or HSV-2 virions resulted in the synthesis of DNA which was resistant to cleavage by restriction endonuclease (Endo R.) HindIII. Defective DNAs of type 1 and 2 were observed to have similar biphasic renaturation kinetics and to have kinetic complexities of approximately 13% of the parental standard genome. Not more than 40% of the sequences found in defective HSV-2 DNA were found to be homologous to defective HSV-1 sequences. While an overproduction of an early polypeptide, VP175, was observed in cells infected with defective HSV-1, no overproduction of any polypeptide equivalent to VP175 was observed in cells infected with defective HSV-2. The results suggest that although the defective DNAs of HSV-1 and HSV-2 have some common physicochemical properties, their base sequences and genomic expression in the infected cell are different.     

Human Cytomegalovirus and Human Herpesvirus 6 Genes That Transform and Transactivate

This review is an update on the transforming genes of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6). Both viruses have been implicated in the etiology of several human cancers. In particular, HCMV has been associated with cervical carcinoma and adenocarcinomas of the prostate and colon. In vitro transformation studies have established three HCMV morphologic transforming regions (mtr), i.e., mtrI, mtrII, and mtrIII. Of these, only mtrII (UL111A) is retained and expressed in both transformed and tumor-derived cells. The transforming and tumorigenic activities of the mtrII oncogene were localized to an open reading frame (ORF) encoding a 79-amino-acid (aa) protein. Furthermore, mtrII protein bound to the tumor suppressor protein p53 and inhibited its ability to transactivate a p53-responsive promoter. In additional studies, the HCMV immediate-early protein IE86 (IE2; UL122) was found to interact with cell cycle-regulatory proteins such as p53 and Rb. However, IE86 exhibited transforming activity in vitro only in cooperation with adenovirus E1A. HHV-6 is a T-cell-tropic virus associated with AIDS-related and other lymphoid malignancies. In vitro studies identified three transforming fragments, i.e., SalI-L, ZVB70, and ZVH14. Of these, only SalI-L (DR7) was retained in transformed and tumor-derived cells. The transforming and tumorigenic activities of SalI-L have been localized to a 357-aa ORF-1 protein. The ORF-1 protein was expressed in transformed cells and, like HCMV mtrII, bound to p53 and inhibited its ability to transactivate a p53-responsive promoter. HHV-6 has also been proposed to be a cofactor in AIDS because both HHV-6 and human immunodeficiency virus type 1 (HIV-1) have been demonstrated to coinfect human CD4(+) T cells, causing accelerated cytopathic effects. Interestingly, like the transforming proteins of DNA tumor viruses such as simian virus 40 and adenovirus, ORF-1 was also a transactivator and specifically up-regulated the HIV-1 long terminal repeat when cotransfected into CD4(+) T cells. Finally, based on the interactions of HCMV and HHV-6 transforming proteins with tumor suppressor proteins, a scheme is proposed for their role in oncogenesis.     

Tumour production by HSV-2 transformed lines in rats and the varying response to immunosuppression.

Rat embryo cells transformed by two ts mutants of HSV-2 strain HG 52 and also by u.v.-irradiated HSV-2 strain 333 were inoculated into highly inbred host rats, either newborn or which had undergone various immunosuppressive treatments. The latent period before a palpable tumour (a fibrosarcoma) was detected varied directly with the degree of immunosuppression of the host. Transformed cells could form tumours after a latent period of nearly 2 years. All tumours were invasive and in some cases metastatic. The continuing expression of HSV information in 10 tumour cell lines was demonstrated by perinuclear and cytoplasmic staining in immunofluorescence studies using a rat antiserum directed against the early polypeptides of HSV-2 HG 52 infection and a rabbit serum prepared against a 24 h cell infection with HSV-2 HG 52 ts I. Sera from tumour-bearing rats fluoresced the surface of unfixed human or rat embryo cells 4 to 5 h after infection with HSV-2 HG 52. In addition the rabbit antiserum (4740 or 4741) fluoresced the surface of 80% of the tumour cells in culture. Transplanted tumours after 20 passages in vitro and taking up to a year to again form a tumour in a host rat also showed specific HSV cytoplasmic and perinuclear fluorescence in tests with the rat antiserum directed against early polypeptides of HSV-2 lytic infection.     

Detection and typing of HSV-1, HSV-2, CMV and EBV by quadruplex PCR

The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.     

Expression of varying portions of the adenovirus 12 early region 1 in transformed cells affects tumorigenicity and interaction with extracellular matrix components

Seven syngeneic rat cell lines transformed with the EcoRI-C (0 to 16.5 map units), SalI-C (0 to 10.3 map units), HindIII-G (0 to 6.8 map units), or AccI-H (0 to 4.7 map units) DNA fragments of highly oncogenic adenovirus 12 were tested for their tumorigenicity in syngeneic hosts and for their interaction with extracellular matrix components. Three of the cell lines (RFC1, EcoC-3, and SalC) were highly tumorigenic and induced tumors with a short latency period; two cell lines (HindG-2 and AccH-1) were also tumorigenic, but the latency period was significantly longer. The HindG-3 and AccH-4 cell lines were nontumorigenic. Collagen attachment preference was analyzed. All five tumorigenic cell lines showed a striking preference for type IV versus type I collagen in attachment assays. The nontumorigenic cell lines showed no preference for either type of collagen. Immunofluorescence analysis of the glycoprotein attachment factors laminin and fibronectin revealed a positive correlation between tumorigenicity, expression of the adenovirus 12 E1 region, and the amount of cell surface laminin. All of the cell lines displayed similar amounts of fibronectin on their surfaces. A similar correlation was observed with the laminin and fibronectin that was secreted into the culture medium. The highly tumorigenic cell lines secreted the greatest amount of laminin, while the nontumorigenic cell lines secreted the least. All of the cell lines secreted comparable amounts of fibronectin into the medium. The results suggest that preference for type IV collagen as well as the presence of cell surface laminin and its secretion are properties associated with expression of the E1 region of highly oncogenic adenovirus 12 in tumorigenic transformed cells.     

Isolation of v-fms and its human cellular homolog

The integrated form of McDonough FeSV proviral DNA, including cellular flanking sequences, was molecularly cloned from nonproductively transformed Fisher rat cells. Acquired cellular-derived (v-fms) sequences within the cloned proviral DNA were mapped from between 2.6 and 5.5 kb from the 5'LTR. Upon transfection, the cloned proviral DNA was biologically active; it caused induction of the transformed phenotype and the resulting transformed cells expressed the major McDonough FeSV translational product, P170gag-fms at high level. Using a series of molecular probes representing subgenomic regions of the viral v-fms gene, a cosmid library of human lung carcinoma DNA was screened for v-fms homologous sequences. Three cosmid clones containing overlapping v-fms homologous cellular DNA inserts, representing a contiguous region of cellular DNA sequence of approximately 64 kb in length, were isolated. Within this region of human genomic DNA, v-fms homologous sequences are dispersed over a total region of around 32 kb. These represent the entire human cellular homolog of v-fms, are colinear with the viral v-fms transforming gene, and contain a minimum of four intervening sequences. At least 12 regions of highly repetitive DNA sequences have been mapped in close proximity to c-fms coding sequences.     

Conservation of a gene cluster including glycoprotein B in bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1)

A library of subgenomic fragments of bovine herpesvirus type 2 (BHV-2) DNA was constructed in the expression cloning vector lambda gt11 and screened with monoclonal antibodies to the glycoprotein gb BHV-2, which is homologous to glycoprotein gB (gB-1) of herpes simplex virus type 1 (HSV-1). Lambda gt11 clones containing gB BHV-2-specific sequences were used to identify lambda EMBL3 vectors with DNA inserts which contained the complete gB BHV-2 gene. Nucleotide sequencing revealed that the gB BHV-2 gene is highly conserved compared to gB-1. The amino acid sequences and the predicted secondary structures of both glycoproteins are very similar. Two further open reading frames (ORF) in close vicinity to the gene encoding gB BHV-2 showed considerable homology to HSV-1 genes. They code for the major DNA-binding protein (dbp) of BHV-2 and a putative 72-kDa polypeptide. The gene of the latter protein corresponding to ICP18.5 of HSV-1 is interspersed between the ORFs of gB BHV-2 and the dbp of BHV-2. All three genes map in the unique long region of the genome. Their homology and the colinear arrangement compared to HSV-1 indicate a close relationship between the two viruses.     

Investigation of the virulence genes of herpes simplex virus 2 by experimental infection in vivo with defined intertypic recombinants of a virulent HSV-2 X an avirulent HSV-1

For determination of the gene loci for virulence and tropism of herpes simplex virus 2 (HSV-2) intertypic recombinants of HSV-1 strain HFEM and HSV-2 strain 3345 were screened for their pathogenicity in the tree shrew. HSV-1 strain HFEM is not pathogenic in the tree shrew and can be recovered as a latent virus only from the spleen of the animals; but HSV-2 strain 3 345, the other parental virus of the recombinants, was found to be virulent for the tree shrew and colonized the ganglia of latently infected animals. No clinical pictures were observed when the animals were inoculated with HSV recombinants RS1₃, RB2₃, RB2₈, RB2₉, RB2₁₀, RB5₂, RB7₃, and RB7₇. It was found, however, that HSV recombinant RB5₀ is pathogenic for the tree shrew. Studies of the state of viral latency in the animals infected with these recombinants revealed that only HSV recombinant RB5₀ was able to persist as latent virus in the spleen of latently infected animals. The genome of recombinant HSV-RB5₀ contains DNA sequences of HSV-2 strain 3345 with two interruptions at 0.22–0.33 and 0.54–0.59 viral map units. In these regions of HSV-RB5₀, DNA sequences of HSV-strain HFEM at 0.26–0.33 and about 0.57–0.58 viral map units, respectively, had been recombined. The results obtained in this study indicate that the recombinant HSV-RB5₀ acquired the virulent phenotype (v) from parental virus HSV-2 strain 3345 but lost the phenotype for colonizing the ganglia (g^b+). Furthermore, it acquired the phenotype of parental virus HSV-1 strain HFEM for persisting in a latent state only in the spleen (s and g) of the latently infected animals.     

Identification of adenovirus-type 12 gene products involved in transformation and oncogenesis

Primary baby rat kidney cells were transfected with Ad12 DNA fragments EcoRI C (left-terminal 16%) and HindIII G (left-terminal 7.2%), and the resulting transformed cells were established as cell lines. Injection of newborn hamsters with purified Ad12 DNA resulted in the induction of tumors in 2 out of 59 animals. A number of the in vitro transformed cells and a cell line derived from a tumor were found to contain DNA sequences homologous to the fragments used for transfection or tumor induction, and to express virus-specific RNA and T antigens. The cell lines were also studied with respect to their tumorigenicity in nude mice or hamsters. It was found that cells transformed by Ad12 EcoRI C, or by intact DNA or virus, were tumorigenic whereas cells transformed by fragment HindIII G were unable to induce tumors. To correlate this result with the presence or absence of viral gene products, virus-specific T antigens were identified by immunoprecipitation. From lytically infected cells major proteins of 60, 41, 19, 14.5, and 13.5 kD were precipitated. Cells transformed by fragment EcoRI C or intact viral DNA contained proteins of 60, 41, and 19 kD and of 50 and 36 kD. HindIII G-transformed cells contained proteins of 41 and 19 kD. Studies of the T antigens in two-dimensional gels and by tryptic peptide analysis have indicated that two virus-specific 60-kD proteins are expressed in Ad12-infected cells. The major protein probably represents the single-stranded DNA-binding protein encoded by region Ell (Ell-60 kD), while the minor protein represents a region EI-specific T antigen (EI-60 kD) encoded by region EIb. The 41-kD protein is encoded by region EIa and 19 kD by EIb. Our results suggest that expression of the EI-60-kD protein is required for oncogenicity in nude mice, but not for morphological transformation.