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A negative regulatory element present in the human papillomavirus type 16 genome has been characterized. Deletion analysis has localized the 5' end of the element to the late region of the genome at the extreme 3' end of the coding region of the L1 open reading frame, around the L1 stop codon, with the element extending into the L1 3' untranslated region. For the cell lines used, the element's function was independent of cell type, tissue, or species of origin, unlike papillomavirus infection, which is very dependent on such factors. By using an mRNA decay assay, we have determined that polyadenylated RNA containing the element is much less stable than polyadenylated RNA lacking the element. This indicates that the element acts as an mRNA instability element. The significance of A-rich, GU-rich, and AUG-rich sequences for the functioning of this human papillomavirus type 16 instability element is discussed.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

A 3' co-terminus of two early herpes simplex virus type 1 mRNAs has been identified using the nuclease -S1 mapping procedure with cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region 0.56-0.60, are unspliced and are transcribed rightwards on the prototype genome orientation. The position of their 3' ends has been located on the virus DNA sequence and lies downstream from the polyadenylation signal 5'-AATAAA-3'. This hexanucleotide sequence also was present in the complementary DNA strand and was shown to be the polyadenylation signal for a leftwards-transcribed late mRNA. The abundance within the cytoplasm of the 5.0 kb and 1.2 kb mRNAs was investigated. Results indicated that these mRNAs were regulated in concert. It is suggested that sequences at the 3' co-terminus may be involved in their regulation.

Summary Partial denaturation maps of 30 HSV-1 DNA molecules have been obtained using a procedure designed to avoid possible hydrolysis of the DNA at alkalilabile bonds. From the denaturation pattern of the long unique DNA region these molecules were divided into two groups comprised of 16 and 14 molecules. Histogram plots relating the percentage denaturation to position on the DNA for these two groups were aligned in a manner appropriate to the HSV-1 genome model. It was apparent that these groups had the orientation of the long region inverted with respect to each other. Similarly, from the denaturation maps of the short unique region, the molecules were divided into two groups each comprising 15 molecules. Alignment of the histogram plots of these groups indicated that the orientation of the short region was inverted in one group relative to the other. These partial denaturation data confirm the presence of four HSV-1 genome arrangements resulting from the possible combinations of inversions of the two unique DNA regions.

Herpes simplex virus (HSV) is an endemic human virus characterized by latent infection of sensory ganglia, with reactivation leading to peripheral lesions in a proportion of cases. The virus genome (145 500 base pairs) comprises two segments, L and S, bounded by inverted repeat regions of approximately 10500 base pairs (TR L and IR L ) and 6000 base pairs (IR s and TR s ) respectively. A direct terminal repeat of about 300 base pairs is also present in an inverted form at the junction between L and S. Numerous indirect studies have suggested an association of the venereal serotype (HSV-2) with squamous carcinoma of the cervix (Nahmias et al . 1970; Thomas & Rawls 1978). Direct evidence has been notably lacking, but the presence of HSV RNA in cervical neoplasms has recently been reported (Jones et al . 1979; McDougall et al . 1980), with in situ hybridization to tritiated HSV DNA. In collaboration with Dr Frank Sharp of the Department of Midwifery, Glasgow University, we have now completed a major screening of cervical neoplasms by the in situ technique. Patients referred to Dr Sharp’s clinic for colposcopic examination were swabbed to detect intracervical infectious HSV. Punch biopsy samples (approximately 5 mm in section) were taken for pathological examination and categorization as non­-neoplastic cervical intraepithelial neoplasia (c. i. n.) or squamous carcinoma. Parallel punches were snap-frozen and thin sections (7 µm) mounted on glass slides, fixed and then hybridized to in vitro 125 I-labelled HSV-2, Ad2, Ad5 or phage lambda DNA. Biopsies of primary adenocarcinomas of the cervix were treated in the same way. After washing and autoradiography, results are expressed as background-corrected mean grain counts per field (mean of ten fields per sample, 3.6 x 10 –2 mm 2 per field; ca . 20 cells per field).

We have determined the orientation of 4 immediate early (IE) mRNA's on the herpes simplex virus type 1 genome by mapping cDNA's complementary to the 3'-termini of messages. These IE mRNA's are transcribed by a pre-existing cell RNA polymerase, and we propose a model which allows their synthesis from a circular template using a single virus promoter region. The promoter region, which is located in the two repetitive DNA regions which flank the short unique region of the virus genome, may serve to initiate bidirectional transcription of these IE mRNA's.

A transcription unit at the herpes simplex virus (HSV) type 2 transforming region, mtr-2 (map coordinates 0.580-0.625), comprises two early, unspliced mRNAs of 4.5 kb and 1.2 kb which are 3' co-terminal; a region including that specifying the 1.2-kb mRNA has been sequenced. The putative translated portions of these two mRNAs do not overlap and this feature, together with the arrangement of the mRNAs, is similar to the apparently equivalent co-linear HSV-1 locus which however does not transform. A putative stem and loops structure containing a TATA box is located upstream from the 5' terminus of the 1.2-kb mRNA within the translated portion of the 4.5-kb mRNA. Evidence for the generation of this structure by intra-strand reassociation under our hybridisation conditions has been obtained and possibilities are that it may function as a transcription-activated promoter or as an RNA polymerase pause site. A comparison of the equivalent HSV-2 and HSV-1 regions reveals a conserved sequence downstream from the 3' co-terminus which is present at a similar location in many eukaryotic genes (consensus sequence YGTGTTYY). The overall sequence conservation at this transcription unit is high except for regions located at: (1) the untranslated leaders of the 1.2-kb mRNAs; (2) the N termini of the polypeptides specified by the HSV-2/HSV-1 1.2-kb mRNAs; (3) the intergenic region beyond the 3' co termini. Regions (2) and (3) are located within a transforming fragment of HSV-2. The possible significance of these data for HSV-mediated cell transformation is discussed.

Summary 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.

Introduction. Herpesviruses infect a wide range of vertebrates and are important human and animal pathogens: in a number of cases infection persists for very long periods, in a latent form, with occasional episodes of overt typical disease. In addition, several herpesviruses have been shown to be the causative agent of tumour formation in their normal hosts (e.g. Lucke frog tumour virus) or in related species (e.g. herpesvirus saimiri). Indirect evidence exists for the involvement of Epstein-Barr virus and herpes simplex virus type 2 (HSV-2) in human cancers. Thus there exist good reasons for the pursuit and identification of the mechanisms of herpesvirus infection and replication. From the molecular virological standpoint, herpesviruses also offer an important system for study. This large virus has the potential to code for about one hundred genes and the identification of these gene products, their function and their control poses many interesting questions.

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.

Summary The mol. wt. and molar ratios of the Hind III and Hpa I fragments of HSV-1 DNA and the Eco RI fragments of HSV-2 DNA have been determined. Results obtained suggest that DNA isolated from both HSV-1 and HSV-2 consists of molecules with four different sequence arrangements which are present in similar amounts. Our explanation of the cleavage patterns of these four genome arrangements with the different restriction enzymes is presented. Some of the possible implications of these four genome arrangements for genetic recombination are discussed.

Herpes simplex virus type 1 (HSV-1) encodes a ribonucleotide reductase which comprises two polypeptides with sizes of 136,000 (RR1) and 38,000 mol. wt. (RR2). We have determined the entire DNA sequence specifying HSV-1 RR1 and have identified two adjacent open reading frames in varicella-zoster virus (VZV) which have homology to HSV RR1 and RR2; the predicted sizes for the VZV RR1 and RR2 polypeptides are 87,000 and 35,000 mol. wt. respectively. Amino acid comparisons with RR1 and RR2 polypeptides from other organisms indicate that HSV-1 RR1 contains a unique N-terminal domain which is absent from other RR1 polypeptides apart from HSV-2 RR1. These N-terminal amino acid sequences are poorly conserved between HSV-1 and HSV-2 in contrast to the remainder of the protein which shows greater than 90% homology. Polypeptide structural predictions suggest that the HSV-1 N-terminal domain may be separated into two regions, namely, a beta-sheet structure followed by a nonstructured area. Across the remainder of RR1 and RR2, comparisons also reveal blocks of amino acids conserved between the different ribonucleotide reductases, and these may be important for enzyme activity. From predictions on the structure of these conserved blocks, we have proposed that the location of a substrate binding site within RR1 is centered on three conserved glycine residues in a region which is predicted to adopt a beta-sheet/turn/alpha-helical structure; this approximates to the structure for ADP nucleotide binding folds. Finally, we propose that the promoters for the HSV and Epstein-Barr virus (EBV) RR2 transcripts have evolved by separate evolutionary routes.

Summary The 5′ ends of two early herpes simplex virus type 1 mRNAs have been identified by nuclease S1 and exonuclease VII analysis using cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region between map coordinates 0.56 and 0.60, are unspliced and share a 3′ terminus. Genomic DNA at the 5′ ends has been sequenced and the 5′ termini have been located on the virus DNA sequence. The DNA sequence has revealed signals involved in the initiation of transcription of both mRNAs, and the 5′ end of the 1.2 kb mRNA is encoded within the internal sequences of the 5.0 kb mRNA. The probable translational initiation codons for the polypeptides specified by these mRNAs have been identified, and the results indicate that the coding regions of the two mRNAs do not overlap.

Polyadenylated immediate-early transcripts of herpes simplex virus type 1, made in BHK cells infected and maintained in the presence of cycloheximide, have been separated on denaturing agarose gels containing methyl mercuric hydroxide. Three virus-specific mRNA bands of estimated sizes 4.7, 3.0, and 2.0 kilobases (kb) were detected, and these mRNA's were mapped on the virus genome and also used to direct protein synthesis in vitro. The 4.7- and 3.0-kb mRNA's hybridized predominantly to certain DNA fragments which are located in the short and long repetitive regions of the genome, respectively, whereas the 2.0-kb mRNA's mapped to three discrete regions of the virus DNA. In vitro translation of these separated mRNA size classes indicated that the 3.0-kb mRNA specified the synthesis of virus polypeptide Vmw 110, whereas the 2.0-kb mRNA's specified Vmw 68, 63, and 12. The synthesis of small amounts of Vmw 175 was specified by the 4.7-kb mRNA. In contrast with the mRNA's which specify these other immediate-early polypeptides, that specifying Vmw 12 is much larger than required for its coding sequences.