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Lytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from he cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effect on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated teh expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.

A set of polypeptides has previously been described as being immunoprecipitated from tumor cell lysates by the sera of tumor-bearing animals (TBS) or by antisera raised to herpes simplex virus type 2 (HSV-2)-infected cells. These polypeptides were not immunoprecipitated from control cells. The principal polypeptides detected of MW 200, 90 (U90 and L90), 40, and 32 kDa were increased by HSV-2 infection. This communication describes the purification of the 40-kDa protein and its identification by amino acid sequence analysis as being homologous to the mature form of mitochondrial aspartate amino transaminase (mAAT). Two different forms of mAAT were purified and sequenced. One form, of low abundance, was immunoprecipitated by TBS and corresponded to the 40-kDa protein immunoprecipitated from tumor, but not control, cell lysates. Western blotting of the 40-kDa protein immunoprecipitated by TBS showed that it was a form of mAAT found only in tumor cells. The other abundant form reacted in Western blots with antibody to mAAT and was clearly the constitutive enzyme, present in all cells. In general, mAAT was increased up to fourfold after infection with HSV-2, HSV-2 infection also increased mAAT enzyme activity. Northern blotting and quantitative PCR showed that mAAT was induced by HSV-2 at the level of transcription. The specific form of mAAT immunoprecipitated from tumor, but not control, cell lysates could have a role in tumorigenesis, could be a putative tumor cell marker, or could be a target for antitumor drugs. HSV-2 probably induces enzymes of glutamine metabolism because HSV-2 requires glutamine for growth, thus revealing potential new antiviral targets.

Infection of primary rat embryo cells with herpes simplex virus type 2 has previously been reported to produce a dramatic and rapid inhibition of cellular DNA methylation. However, it has neither an immediate effect on S-adenosylmethionine breakdown nor on the relative pool sizes of S-adenosylmethionine and S-adenosylhomocysteine.

A set of polypeptides detected in transformed cells of M(r) values 90,000 (a doublet) 40,000 and 32,000 that are recognised by immunoprecipitation of L-[35S]methionine-labelled tumour cell lysates, with sera from tumour-bearing rats and with antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, were previously reported (Macnab et al., 1985, 1992; Hewitt et al., 1991). These proteins are cell-coded and cannot be precipitated from similarly radiolabelled control cells. Two of these polypeptides are significantly induced by HSV-2 infection (Hewitt et al., 1991; Macnab et al., 1992). This paper further characterises one of these polypeptides, U90, and addresses which properties distinguish the behaviour of U90 in tumour cells, whether there is an equivalent in control cells and whether U90 can be induced without HSV replication. U90 can be immunoprecipitated from radiolabelled human cells which are capable of extended passage in culture, as well as from human tumour cells, rodent tumour cells and cells of different lineages, e.g. epithelial, fibroblastic and lymphoid. Purification of U90 and the subsequent production of monospecific antibodies revealed, by western blotting, a homologue of 90 kDa in control cells. Western blotting shows that HSV can increase the amount of the U90 homologue in these normal cells. The absence of an immunoprecipitate of the U90 homologue from control cells is a result of the very short half-life (i.e. high turnover) of the protein in these cells (32.9 minutes) as opposed to tumour cells (about 13 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Normal and abnormal biopsies of the uterine cervix, to a total of 124 samples, have been analysed for the detection of oestrogen receptor (ER) mRNA. The tough fibrous nature of the cervix proved very resistant to disaggregation in the first instance and subsequently, it was difficult to extract good high molecular weight message. This necessitated a systematic study of methodological technique, including two methods of tissue disaggregation and five techniques of extraction of ER mRNA, which in total involved the use of 124 cervical biopsies. The most successful method involved chopping the tissue, then digesting the cells with proteinase K and extracting the nucleic acids in salt and sodium dodecyl sulphate. Using the perfected technique, 16 cervical biopsies obtained at serial intervals from four women did not show any differences in ER mRNA in cervical biopsies either in the presence of oral contraception or histological abnormality. The successful method described will prove valuable for the detection of ER message in human tumours and other tissues of similar nature.

Anti-sera raised against HSV-2-infected cells (WI) and the sera of animals bearing tumours (TBS) to HSV-2 transformed cells contain antibodies to a set of tumour-specific cell-coded polypeptides. The specificity of these polypeptides for tumour cells is monitored by the ability of [35S]-L-methionine labelled proteins to be immunoprecipitated by these anti-sera, in contrast to control cells from which the polypeptides are not precipitated. The polypeptides which share an epitope and are co-precipitated are of MWs 90,000 (a doublet), 40,000 and 32,000. The upper 90,000-MW polypeptide (U90) is induced by HSV-2 infection. This communication deals with the 40,000-MW polypeptide which was shown to be immunoprecipitated by TBS and a monoclonal antibody (MAb) raised to the DNA-binding proteins of HSV-2-infected cells. Immunological and biochemical studies reveal that the 40,000-MW protein which is immunoprecipitated comprises more than one polypeptide, and that the proteins may need to interact to produce the peptide pattern specific for the tumour form of the immunoprecipitated 40,000-MW protein. WI antisera and TBS both recognise antigens specific for tumour cells in sections of cervical-carcinoma tissue. Sera from patients with cancer of the cervix contain antibodies to a cell-coded polypeptide of MW 40,000, which by peptide analysis is indistinguishable from the 40,000-MW polypeptide induced by HSV-2 infection and immunoprecipitated by WI and TBS.

A tumour-specific polypeptide designated U90 is one of a set of polypeptides which are encoded by the host cell and are specific for the transformed cell state, being immunoprecipitated by the sera of tumour-bearing animals. The interest in these tumour-specific polypeptides centres on the finding that they are also recognized by antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, implying some role for HSV-2 in tumorigenesis. The peptide map of HSV-2-induced U90 is indistinguishable from that of U90 present in uninfected tumour cells, including mouse cells transformed by human papillomavirus type 16. In tumour cells, U90 is located principally in the plasma membrane fraction and cannot be induced by heat shock, glucose starvation, or treatment with tunicamycin or calcium ionophore. U90 is not related to either the heat shock protein of Mr 90,000 (HSP90) or the glucose-related polypeptide of Mr 94,000 (GRP94) as determined by peptide mapping and the use of monospecific, monoclonal and antipeptide antibodies. This suggests that U90 is a novel transformation-specific protein which can be induced by infection with HSV-2.

Objective: To observe in serial fashion the histological changes of human papilloma virus (HPV) infection of the cervix and to correlate these with the detection of HPV genomes., Design: A prospective longitudinal study to perform colposcopy and at 0, 9 and 18 months to obtain a smear, a biopsy for histology and HPV-16 genome detection by DNA/DNA hybridization., Setting: Glasgow Family Planning Centre., Subjects: Eighty-two women recruited on the basis of a routine smear showing viral change without dyskaryosis., Interventions: Women found to have CIN 3 were treated with laser., Main Outcome Measures: Cytology and cervical biopsy results and HPV-16 DNA detection by Southern blotting obtained serially from individual subjects., Results: Of 82 women recruited, 10% were positive for HPV-16 detection by Southern blotting. Detection of HPV-16 genomes did not correlate with either CIN or viral infection assessed histologically and serial hybridizations in a given individual showed fluctuation in HPV-16 detection. Polymerase chain reaction in a subset of samples revealed 46% positive for HPV DNA., Conclusion: No clear correlation exists between HPV genome detection and the histological appearance. HPV-16 genome detection alone may not be a useful predictor of precancerous progression.

A number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences. One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell. Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences. Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization. All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement. The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration. The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences. Possible consequences of this deletion for viral replication and transcription are discussed.

Four independent isolated lines transformed by ts mutant or sheared DNA of herpes simplex virus types 1 and 2 and one tumor cell line were characterized according to their behavior in assays of aggregation, growth in low serum, doubling times, saturation density, agglutination by lectins, uptake of metabolites, and production of plasminogen activator. The criteria used to select the transformed cells used in this study were the formation of altered foci of cells followed by the formation of a continuous cell line, the continuing expression of herpes simplex virus-specific antigens, and the tumorigenicity of the transformed cell lines. The results indicate that, whereas the one herpes simplex virus type 2-transformed cell line investigated showed many of the transformation parameters accepted as typical of cells transformed by the papovaviruses, the three herpes simplex virus type 1-transformed cell lines do not share or express many of these transformation parameters.

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.

Biopsy samples from 27 patients referred to a colposcopy clinic in Glasgow for cervical abnormalities were assessed for the relations among colposcopic appearances, cytological and histological diagnosis, expression of papillomavirus antigen, and the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 deoxyribonucleic acid (DNA) sequences. Specimens were from colposcopically abnormal areas of the transformation zone and from colposcopically apparently normal areas of the zone in the same patients (paired matched internal control tissue). All 27 women referred for abnormal smears had colposcopic abnormalities. HPV-16 or 18 DNA sequences were detected in 20 of the 27 colposcopically abnormal biopsy samples and 13 of the 27 paired normal samples. Twelve samples of colposcopically normal tissue contained histological evidence of viral infection but only four of these contained HPV DNA sequences. The other nine samples of colposcopically normal tissue which contained HPV DNA sequences were, however, histologically apparently normal. HPV-6 and 11 were not detected. Integration of the HPV-16 genome into the host chromosome was indicated in both cervical intraepithelial neoplasia and control tissues. In two thirds of the HPV DNA positive samples the histological grade was classed as normal, viral atypia, or cervical intraepithelial neoplasia grade 1. Papillomavirus antigen was detected in only six of the abnormal and three of the normal biopsy samples, and HPV DNA was detected in all of these. The detection of HPV DNA correlates well with a combination of histological and cytological evidence of viral infection (20 of 22 cases in this series). A poor correlation between the site on the cervix of histologically confirmed colposcopic abnormality and the presence of HPV DNA sequences implies that a cofactor other than HPV is required for preneoplastic disease to develop. A separate study in two further sets of biopsy samples examined the state of HPV DNA alone. The sets were (a) 43 samples from cervical intraepithelial neoplasia and nine external controls and (b) 155 samples from cervical intraepithelial neoplasia, cervical cancer, vulval intraepithelial neoplasia, and vulval cancer and external controls. HPV-11 was found in only two (4.7%) of the 43 specimens from cervical intraepithelial neoplasia, whereas HPV-16 was found in 90 (58%) of the other 155 specimens. These results also suggest that HPV subtype is subject to geographical location rather than being an indicator of severity of the lesion or of prognosis.

Infection of estrogen-responsive cells (MCF-7) with herpes simplex virus type 1 or 2 stimulates expression of the estrogen receptor message. Experiments on infection with the mutant virus, tsK, together with transfection studies implicate the virion protein, Vmw65, in the response. Cellular protein synthesis is essential for estrogen receptor mRNA expression.

A total of 143 consecutive patients with abnormal cervical cytology was examined at a large colposcopy clinic in Glasgow. Each patient had paired biopsies from normal and abnormal cervical epithelium examined both histologically and by immunoperoxidase staining for human papillomavirus (HPV) infection. More than 71% of the abnormal biopsies and 39% of the normal paired biopsies had histological evidence of HPV infection. Cytological evidence of HPV infection was seen in 38.5% of cervical smears. Immunocytochemistry revealed HPV antigen in 22% of the abnormal biopsies and in 4.2% of the 'normal' biopsies. The presence of HPV infection in colposcopically normal cervical tissue both inside and outside the transformation zone may help to explain why current methods for treatment of cervical HPV infection are often unsuccessful.

A rat cell line, RE1, oncogenically transformed by a herpes simplex virus type 2 ts mutant (ts 1), has been demonstrated to contain herpes simplex virus type 2-specific thymidine kinase activity, as have two of four tumors induced in rats by inoculation of these transformed cells. A high proportion of sera from tumor-bearing rats (5 of 11) have detectable antibody against herpes simplex virus thymidine kinase, and there is a correlation between enzyme activity in a tumor and antibody in "tumor sera." A proportion of tumor sera possess neutralizing activity for herpes simplex virus infectivity. Immunfluorescence studies indicate that the transformed cells express antigens which are probably induced early in herpes simplex virus type 2-productive infection.

Herpes simplex virus (HSV) and human cytomegalovirus (HCMV) are candidates for the induction of premalignant or malignant disease. Morphological transformation studies have failed to demonstrate a viral oncogene, a virus-coded transforming protein or any sequence of DNA that uniquely transforms cells according to one-hit kinetics. Thus the mechanism of transformation is complex. The transformed cells are, however, all oncogenic in the host animal and in immunocompetent mice. Direct evidence for the presence of these viruses in human genital tumours is the finding that a small proportion (about 10%) retain fragments of virus DNA from different regions of the virus genomes. In contrast human papillomavirus (HPV) is strongly associated with genital neoplasia, being present in over 80% of tumours. However, HPV can also be detected in histologically normal tissue. The most persuasive roles for HSV and HCMV in human tumourigenesis are as mutagens, as activators of cellular transcription or in switching on the synthesis of host cell proteins not normally expressed in untransformed cells. In these roles the prospects of further defining roles for HSV and HCMV in the multistage process of oncogenic transformation are good.

A rat tumour induced by cells transformed with the sheared DNA of herpes simplex virus (HSV) type 1 HFEM alpha (RE2A) was injected with the intertypic virus HSV-2 HG52 ts 1. Separate plaques, isolated from cocultivation of excised tumour tissue with susceptible cells, yielded virus the DNA of which had the restriction enzyme profile either of the injected HSV-2 virus or that of the HSV-1 virus, originally used to transform the cells. No evidence of in vivo recombination was detected. In Hooded Lister rats, HSV may have the ability to remain in a latent or non-replicating state in fibroblasts.

The chromosomes of six rat embryo cell lines transformed with herpes simplex virus (HSV) temperature-sensitive (ts) mutants were examined at different passages of in vitro cultivation. Two cell lines were predominantly diploid, one cell line was hyperdiploid, one cell line was pseudodiploid, and two cell lines were hypotetraploid. In near-diploid cell lines chromosome No. 9 was most frequently involved in chromosome changes. All three cell lines derived from tumors obtained after one transplantation of HSV-transformed cells into baby rats were pseudodiploid, but each had different marker chromosomes. Chromosome No. 15 was involved in the formation of two out of four marker chromosomes. Four cell lines derived from tumors developing after two and three transplantations were hypodiploid and showed large chromosome variation. The occurrence of 25 marker chromosomes in three tumor-derived cell lines resulted in gains in parts from chromosomes No. 2, 6, and 7. One marker chromosome had a homogeneously faintly stained region. Chromosomes No. 2, 3, 7, and 12 were more frequently involved in the formation of marker chromosomes. No chromosome change was found to be specifically associated with HSV-induced transformation of rat cells, but chromosome changes in tumor-derived cell lines may provide selective advantage for survival and autonomous growth in the host animal.

Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence.

Punch biopsies were taken from patients with cervical intraepithelial neoplasia and the DNA was extracted and examined for sequences which hybridized to human cytomegalovirus DNA by Southern blotting analysis. Two biopsies out of 43 were found to contain DNA which hybridized to human cytomegalovirus DNA. In one biopsy DNA sequences were detected which contained four restriction sites colinear with those of the prototype strain AD169. Evidence of rearranged viral DNA sequences was also found.

Both cervical cancer and its precancerous state cervical intraepithelial neoplasia (CIN) have the characteristics of being sexually transmitted. Formerly herpes simplex virus (HSV) but more recently human papillomavirus (HPV) which both infect the cervix have been implicated in causation. The role of these viruses as possible initiators of cancer or as potential cofactors of cocarcinogens is discussed at the molecular level within the context of the disease process.

LMTK-cells infected with u.v.-irradiated herpes simplex virus type 1 have been selected for the presence of the enzyme thymidine kinase. These cells have an altered morphology compared to the control cells and contain herpes-specific antigens in their cytoplasm. The thymidine kinase activity present in these cells has been shown, on the basis of a number of biochemical properties, to be identical to the herpes virus specified deoxypyrimidine kinase found during lytic infection of this virus. In addition it has been possible to detect herpes simplex-specific RNA sequences in the transformed cells and this occurs in both the polyadenylated and non-polyadenylated cytoplasmic and nuclear fractions.

To study the association of human papillomavirus (HPV) and herpes simplex virus (HSV) with genital cancer, we collected specimens of cervical, vulvar, endometrial, and vaginal tumors at the time of operation in patients with cancer. In some patients, matched internal-control (histologically normal) tissue was also collected. DNA extracted from the tissue was probed with radiolabeled HPV type 16 DNA, HPV type 18 DNA, and cloned fragments of HSV type 2 DNA. Hybridization to the HindIIIa clone of HSV-2 was detected in only 1 cervical tumor and 1 vulvar tumor (9 percent) among the 22 tumors tested. However, DNA sequences hybridizing to HPV-16 were detected in 21 of 25 tumors (84 percent) and in 8 of 11 (73 percent) of the DNA samples from clinically and histologically normal, paired, internal-control tissues from the patients with cancer. HPV-16 DNA was found in one of nine normal cervixes (11 percent) of women without genital neoplastic disease or abnormal cytology. HPV-18 DNA was detected in only 2 of 24 tumors (8 percent), 1 cervical and 1 vulvar. Our results show a strong association between the presence of HPV-16 genomes and genital tumors and between HPV-16 genomes and histologically normal tissue within 2 to 5 cm of the tumors. The implications of these findings remain to be explored.

Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

Nonneoplastic and neoplastic cervical biopsy specimens were examined by in situ hybridization to 125I-labeled DNA of herpes simplex virus (HSV), adenovirus, and bacteriophage lambda DNA's, and quantitative hybridization data were obtained using a Video Image Analyser. HSV-specific RNA was detected in 72% of cervical intraepithelial neoplasia, 60% of squamous cervical carcinomas, 2% of nonneoplastic cervices, and 9% of primary adenocarcinomas of the cervix. None of the tissues gave positive hybridization with adenovirus or lambda DNA probes. In paired biopsies of cervical intraepithelial neoplasia and nonneoplastic epithelium from 29 individuals, HSV-specific RNA was detected only in the epithelium of the neoplastic sample and not in the nonneoplastic control. Infectious HSV-2 was isolated from a low proportion (2%) of both ectocervical swabs and cell-free tissue extracts of patients examined, suggesting that the HSV-specific RNA detected in squamous cell neoplasms was not due to overt infections.

We have determined the herpes simplex virus (HSV) type 2 DNA sequences responsible for the initiation of morphological transformation and have investigated the retention and expression of these sequences in morphologically transformed cells and in tumours derived from these cells. All the transformed cells analysed were selected by a focus formation assay and are oncogenic in the inbred host rat. Cloned HindIII and Bg/II fragments from the HSV-2 genome were assayed for the ability to initiate morphological transformation of rat embryo cells. Only the HindIII a (map units 0.52 to 0.72) and the Bg/II n (0.582 to 0.612) clones gave transformed foci. This shows that the Bg/II n region is responsible for initiation of transformation. Southern blot analysis of DNA extracted from these transformed cells and from tumours derived from these transformed cells revealed that neither the Bg/II n fragment nor fragments of 500 bp mapping within it are detected at the level of one copy per cell and therefore need not be retained in the cell to maintain the oncogenic phenotype. In addition there was no evidence of expression of the HSV-specified ribonucleotide reductase activity which is partially encoded within the Bg/II n fragment of HSV-2. We also analysed DNA from rat embryo cells transformed by ts mutants of HSV-2 (HG52) or HSV-1 (HFEM or 17) at non-permissive temperature or by virus at supraoptimal temperature or by sheared virus DNA and DNA from tumours derived from lines of these transformed cells. In addition, we cloned both transformed and tumour cell lines and analysed these similarly. In no case could we detect HSV DNA sequences at the level of one copy per cell.

DNA extracted from eight human cervical carcinomas, one lymph node metastasis and related control tissue was examined for the presence of herpes simplex virus (HSV) DNA sequences. Southern blot transfers of tumour and control DNA were hybridised with radioactively labelled cloned probes representing 70% of the HSV-2 genome. Specific hybridisation to HSV DNA sequences was observed in one of eight carcinoma tissues analysed. Hybridisation of HSV-2 DNA probes to BamHI and XhoI restriction enzyme fragments of tumour cell DNA which co-migrated with authentic HSV-2 viral fragments identified co-linear HSV-2 DNA sequences comprising 3% of the HSV-2 genome, between map coordinates 0.582 and 0.612. The remaining eight tumour and all control tissues analysed, showed no specific hybridisation to any of the probes used at levels of sensitivity which would detect 0.5 copies/cell of HSV-2 DNA restriction fragments of 2 kb or greater.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.

Two rat embryo cell lines transformed by herpes simplex virus (HSV) and four tumor cell lines derived from inoculation of HSV-transformed cells formed rosettes after treatment with rabbit or human anti-HSV IgG and sheep red blood cells coated with protein A of Staphylococcus aureus, Cowan I strain. The frequency of rosette-forming cells after treatment with rabbit anti-HSV IgG at a concentration of 0.01 mg/10(6) cells varied between 41.6 and 73% depending on the cell line. Pretreatment of HSV-transformed cells with rabbit anti-HSV IgG Fab prevented rosette formation after treatment with rabbit or human anti-HSV IgG. Trypsin digestion of transformed cells reduced significantly the proportion of rosettes formed with human or rabbit anti-HSV IgG. The microscopic visualization of rosette formation on HSV-transformed cells is consistent with the expression of HSV-specific antigens on the cell surface.

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.

We have prepared EMBL3 libraries of DNA extracted from the cervix of a patient with cervical intraepithelial neoplasia (CIN) and isolated seven recombinant clones containing sequences that hybridize to human cytomegalovirus (HCMV) DNA. Restriction analysis of one clone with BamHI and SalI endonucleases revealed that the insert DNA showed a high degree of homology to the HCMV Ad169 genome over the region between the HindIII K/E site and the SalI site located within the BamHI P fragment. The HCMV insert in the CIN clone is integrated and flanked by cellular sequences. The major immediate early gene that encodes a polypeptide of approximately 69 kD was found to be conserved in the CIN clone. Transfection of clones encoding the immediate early region of HCMV resulted in cells that were positive in immunofluorescence studies with two monoclonal antibodies directed against the HCMV 69 kD immediate early polypeptide. Infection of human ectocervical cells with HCMV Ad169 revealed that they could express the 69 kD polypeptide encoded by the immediate early gene but could not replicate the virus, whereas HCMV was able to replicate productively in cultured endocervical cells. HCMV has been shown to activate endogenous retroviruses and also to transcriptionally activate the long terminal repeat of human immunodeficiency virus. Activation of virus and cellular genes by HCMV may be a means by which this virus is involved in the multistage process of oncogenesis and/or the activation of latent infections.

Human cytomegalovirus (HCMV) is a sexually transmitted virus which has been implicated in both cervical cancer and its precancerous state, cervical intraepithelial neoplasia (CIN). The evidence for this and the current possible mechanisms by which HCMV could be involved are discussed at the molecular level.