SUMMARYFive cell lines, SKG-I, SKG-II, SKG-IIIb, QG-U and QG-H derived from cervical carcinomas of Japanese patients, were examined for the presence of human papillomavirus (HPV) DNA and the expression of viral mRNA. The DNA of HPV type 16 was shown to be linked covalently with SKG-IIIb, QG-U and QG-H cell DNA, and HPV 18 DNA with SKG-I and SKG-II cell DNA. Although different regions of the HPV genome were integrated in these cell lines, the non-coding region and an early region including the E6 and E7 open reading frames (ORFs) were conserved in all cell lines. The complete genome of HPV 16 was found in QG-H cells by digestion of the DNA with a single-cut restriction enzyme. The other early region ORFs E 1, E2, E4 and E5 were interrupted by flanking host cell DNA, suggesting that the integration into host cell DNA occurs preferentially in this region. HPV-specific mRNA species were detected in all five cell lines. In the three cell lines containing the HPV 16 genome, mRNAs hybridized with the early region of the genome, covering the entire E6 and E7 ORFs and a minor part of the E10RF, although the amount and size of the major mRNAs varied in these cell lines. These mRNAs did not hybridize with the late region of the HPV genome containing the LI and L20RFs. In SKG-II, SKG-IIIb and QG-H cells we also detected c-myc and c-Ha-ras mRNA expression at about nine times the level of that in normal cells.The human papillomaviruses (HPVs) induce epithelial proliferative lesions of skin or mucosa and have been classified into more than 40 types on the basis of their DNA sequence homology.

Polyacrylamide gel analysis of the structural proteins of African and Asian strains of Chikungunya virus, an alphavirus, showed that both strains contain three structural proteins: glycosylated El and E2, embedded in the viral envelope, and a nonglycosylated nucleocapsid protein. In pulse-chase experiments the precursor protein PE2 was chased into glycoprotein E2, which migrated slightly faster than did glycoprotein El. The third Chikungunya glycoprotein, E3, was not associated with mature virions but was released into culture fluids. With glycoproteins El and E2, separated by glass wool column chromatography, it was shown that hemagglutinating activity is associated with glycoprotein El.

The human papillomavirus type 16 E6 protein exerts a transforming activity through inactivation of tumor suppressor p53. Recently E6 has been shown to have additional transforming activities independent of p53. E6 is able to transactivate or repress several specific viral promoters. However, underlying molecular mechanisms and cellular target genes for the activity are not well understood. Using a differential hybridization technique, we identified the prothymosin alpha gene as a cellular target of E6 transactivation. E6 was able to transactivate the prothymosin alpha promoter in H358 cells lacking p53 and in C33A cells harboring a mutant p53 allele. Disruption of the E-box in intron 1 of the prothymosin alpha promoter abolished the responsiveness to E6. Then we determined if E6 up-regulates the expression of Myc, by which the prothymosin alpha promoter is transactivated through the E-box. We found that E6 is also able to transactivate the c-myc promoter in H358 cells and in C33A cells. These results suggest that E6 is able to transactivate the c-myc promoter independently of p53, and that the prothymosin alpha promoter is subsequently transactivated by Myc.

SUMMARYThe presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23~ of mild dysplasias, 329/o of moderate dysplasias, 55 ~ of severe dysplasias and 62 ~ of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 869/oo of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.Human papillomavirus (HPV) type 16 and type 18 DNAs are frequently found in biopsies from precancerous and malignant cervical lesions (Boshart et al., 1984;Crum et al., 1985;Lehn et al., 1985;Tomita et al., 1986), with HPV 16 DNA being more common than HPV 18 DNA in these lesions (Diirst et al., 1983 ;Boshart et al., 1984;Yoshikawa et al., 1985). HPV 16 DNA has been cloned from an invasive cervical carcinoma (Dtirst et al., 1983) and the complete nucleotide sequence was determined (Seedorf et al., 1985). Recently, Diirst et al. (1985) reported the physical state of HPV 16 DNA in some malignant turnouts and showed that the integration of HPV 16 genome into the host cell DNA occurs with a head-to-tail viral genome arrangement. Their report includes a discussion of HPV integration in connection with the causative event in malignant transformation.Dysplasias are considered to be precancerous lesions and are classified as mild, moderate and severe (Koss, 1978). In order to see whether the integration of HPV 16 DNA into host cell DNA occurs in precancerous lesions, biopsy samples from mild, moderate and severe dysplasias and from cervical cancers were screened for the presence of HPV 16 DNA by dot blot hybridization. Virus-specific restriction fragments were analysed by Southern blot hybridization. We report here the detection of submolar fragments, which might be virus-cell junction sequences, in most dysplasias and carcinomas in situ that harbour HPV 16 DNA.Biopsy samples were collected under colposcopy and kept at -70 °C. High molecular weight DNA was extracted and purified from samples that had been histopathologically confirmed (Tomita et al., 1986). For dot blot hybridization, about 7.5 p.g of the purified DNA was denatured, neutralized, then spotted onto a nitrocellulose filter and hybridized with 32p-labelled cloned HPV 16 DNA in 6 x SSC at 68 °C for 24 h. The specific activity of the HPV 16 DNA was 108 to 2 x 108 c.p.m./~tg. The filter was washed extensively with 0

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Human papillomavirus type 16 (HPV16) genome DNA and its transcripts in biopsied cervical neoplasias were analyzed by simultaneous extraction of DNA and RNA from one biopsied sample. Southern blot analysis revealed that 5 of 20 cervical intraepithelial neoplasias (CINs) contained HPV16 DNAs existing primarily as episomes and two of seven invasive carcinomas harbored HPV16 genome sequences integrated into the host DNA. Northern (RNA) blot analysis showed that the HPV16 genome sequences were transcriptionally active in the five CINs, as well as in the two invasive carcinomas. The pattern of HPV16-specific transcripts in the CINs was uniform, and the major transcripts were 4.2, 2.2, 1.6, and 1.4 kilobases in size. However, the pattern of HPV16-specific transcripts in the invasive carcinomas was variable and different from that in CINs, suggesting that the alteration of transcriptional pattern might play a key role in the development of malignancy.

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