Genomic diversity of human papillomavirus genotype 53 in an ethnogeographically closed cohort of white European women

Human papillomavirus (HPV) genotype 53 is classified taxonomically in alpha HPV genus-species 6, together with HPV-30, HPV-56, and HPV-66 and is considered to be one of three "probable high-risk" HPV genotypes. Recent worldwide comparison of 44 isolates of HPV-53 showed the existence of nine long control region (LCR) genomic variants, which formed a phylogenetic tree with two deep dichotomic branches. In order to investigate further the genomic diversity of HPV-53, a total of 94 isolates of HPV-53 obtained from an ethnogeographically closed cohort of 70 white European women was analyzed. The identification and characterization of HPV-53 genomic variants was based on analysis of three different HPV genomic regions: LCR, E6 and E7. A higher genomic diversity of HPV-53 was identified in the ethnogeographically closed cohort of white European women than has been reported previously on isolates collected worldwide. Altogether, 19 HPV-53 genomic variants, composed of 13 LCR, 13 E6, and 5 E7 genomic variants, were identified. Eleven out of 13 LCR, all E6, and four out of five E7 genomic variants were described for the first time. The present study confirmed dichotomic phylogeny of HPV-53 described previously and, in addition, showed for the first time that after a dichotomic split, both groups of HPV-53 genomic variants formed star-like phylogenetic clusters. In women with persistent HPV-53 infection, HPV-53 genomic variants remained unchanged for up to 51 months. In rare cases, infection with multiple HPV-53 genomic variants is possible. Taking into account the results of this and previous studies, at least 26 different HPV-53 genomic variants exist today.     

Identification of a 450-bp region of human papillomavirus type 1 that promotes episomal replication in Saccharomyces cerevisiae

Human papillomaviruses (HPVs) replicate as nuclear plasmids in infected cells. Since the DNA replication machinery is generally conserved between humans and Saccharomyces cerevisiae, we studied whether HPV-1 DNA can replicate in yeast. Plasmids containing a selectable marker (with or without a yeast centromere) and either the full-length HPV-1 genome or various regions of the viral long control region (LCR) and the 3' end of the L1 gene were introduced into S. cerevisiae and their ability to replicate episomally was investigated. Our results show that HPV-1 sequences promote episomal replication of plasmids although the yeast centromere is required for plasmid retention. We have mapped the autonomously replicating sequence activity of HPV-1 DNA to a 450 base-pair sequence (HPV-1 nt 6783-7232) that includes 293 nucleotides from the 5' region of the viral LCR and 157 nucleotides from the 3' end of the L1 gene. The HPV-1 ARS does not include the binding sites for the viral E1 and E2 proteins, and these proteins are dispensable for replication in S. cerevisiae.     

Polymorphism of human papillomavirus type 31 isolates infecting the genital tract of HIV-seropositive and HIV-seronegative women at risk for HIV infection

The genomic polymorphism of high-risk human papillomavirus (HPV) for types other than 16 has not been extensively described. We describe here the genomic polymorphism of high-risk HPV type 31 in 79 women (62 HIV-seropositive, 17 HIV-seronegative) by PCR-sequencing of the long control region (LCR), E6 and E7. LCR polymorphism was generated by 25 (6.4%) single-nucleotide variations over 391 bases. Each variant compared to the prototype contained from 2 to 13 variations (mean of 9.4 +/- 3.3, median of 10). Considering the number of variation sites in each region of HPV genome, the LCR was more variable than E6 (13 over 496 nucleotide (nt), P=0.03) and E7 (9 over 296 nt, P=0.03). Non-synonymous nucleotide variations were found in 31 (75.6%) of 41 isolates and were observed at six positions in E6. Each of the 8 HPV-31 E7 variants contained from 2 to 5 mutations (mean of 4.29 +/- 1.11, median of 5) compared to the prototype. Three non-synonymous E6 and E7 variations were within cysteine arrays. The LCR prototype was significantly over-represented in Caucasian women (14 (25%) of 56) compared to women of African descent (0 (0%) of 15 women, P=0.03). Four (23.5%) of 17 women with persistent versus 6 (25.0%) of 24 women with transient infections were infected by the prototype (P=1.00). HPV-31 LCR was more polymorphic than oncogenes and was associated with ethnicity.     

Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region.

Homologous, subgenomic fragments of the viral LCR and E6/E7 transforming genes of HPV-18 and HPV-16 were amplified from several primary cervical, penile, and vulvar tumors and cloned into a pUC-18-derived vector. When assayed by a quantitative transformation assay using primary human keratinocytes, the subgenomic regions of HPV-16 and HPV-18 exhibited transforming activities similar to that of the full-length, prototype HPV genomes. More importantly, the HPV-18 LCR-E6-E7 region was approximately 10- to 50-fold more active than that of HPV-16. These studies demonstrate (1) that the transforming activity differences previously observed between prototype HPV-16 and HPV-18 map to the LCR-E6-E7 region, and (2) that individual and independent isolates of HPV-16 and HPV-18 exhibit consistent differences in transforming potential, even when isolated from different anatomic sites.     

Prediction of conserved microRNAs from skin and mucosal human papillomaviruses

Eight human papillomavirus (HPV) types including four cutaneous HPV types (HPV-5, HPV-8, HPV-20 and HPV-38) and four mucosal HPV types (HPV-6, HPV-11, HPV-16 and HPV-18) were selected for this miRNA study. Pre-miRNAs were predicted using a computer programme, and the conserved mature miRNAs were compared to currently known miRNAs. Predicted HPV miRNAs related to miR-466, -467 and -669 were common and specific to the mucosal HPV types. Northern blot hybridization confirmed a predicted miRNA in HPV-positive cervical cancer cell lines encoded by mucosal HPVs. HPV-38 was predicted to express an miRNA conserved to human let-7a and the expression of let-7a, in HPV-38-positive non-melanoma skin cancer (NMSC) biopsies was 10-fold higher than those with HPV-positive (for other types except HPV-38) and HPV-negative NMSCs, suggesting that let-7a expression might be related to HPV-38 infection. Potential gene targets of the predicted miRNA that may aid HPV in infection and pathogenesis were also analysed.     

Human papillomavirus type 16 variants isolated from vulvar Bowenoid papulosis

Tissues from two cases of Bowenoid papulosis of the vulva were found to contain human papillomavirus (HPV) 16 DNA by Southern blot hybridization. Analysis of the hybridization pattern revealed differences in a restriction fragment of one specimen as compared to the HPV 16 DNA prototype. To investigate if these differences could interfere with the expression of such oncogenic viral genomes, the corresponding DNA fragments were cloned and further analyzed. After amplification by PCR and DNA sequencing, a 213 base pairs duplication was mapped in the long control region (LCR) of this HPV 16 variant. One single PCR fragment was obtained from the other Bowenoid papulosis, which is identical in size with the same region in the HPV-16 prototype. The duplication in the HPV-16 LCR analyzed in this study maps upstream of a region containing several regulatory elements.     

Prevalence and stability of antibodies to 37 human papillomavirus types — A population-based longitudinal study

Information about serostability of cutaneous HPV types over time is very limited. We investigated seroprevalence and serostability of 37 different HPV types over 4½ years in an Australian population-based study. Sera and data were analyzed for 390 people who had never been diagnosed with SCC and had blood collected in 1992, 1993 and 1996.Eighty-six percent of participants were seropositive to at least one of the 37 HPV types at baseline. HPV-4 was the type with the highest seroprevalence (41%), followed by HPV-38 and HPV-8 (both 33%). Over 90% of people retained their baseline serostatus during the 4½ year follow-up. Highest serostability was observed for HPV-88 (99.7% stayed seropositive or seronegative), while HPV-65 was least stable with 17% altering their serostatus during follow-up.Seroprevalence to cutaneous HPV types are relatively stable over time, and a single measure can be used as a reasonable marker of long-term antibody status.     

Mucosally-derived HPV-40 can infect both human genital foreskin and cutaneous hand skin tissues grafted into athymic mice

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.     

Human papillomavirus type-16 variants in Quechua aboriginals from Argentina

Cervical carcinoma is the leading cause of cancer death in Quechua indians from Jujuy (northwestern Argentina). To determine the prevalence of HPV-16 variants, 106 HPV-16 positive cervical samples were studied, including 33 low-grade squamous intraepithelial lesions (LSIL), 28 high-grade squamous intraepithelial lesions (HSIL), 9 invasive cervical cancer (ICC), and 36 samples from women with normal colposcopy and cytology. HPV genome variability was examined in the L1 and E6 genes by PCR-hybridization. In a subset of 20 samples, a LCR fragment was also analyzed by PCR-sequencing. Most variants belonged to the European branch with subtle differences that depended on the viral gene fragment studied. Only about 10% of the specimens had non-European variants, including eight Asian-American, two Asian, and one North-American-1. E6 gene analysis revealed that 43% of the samples were identical to HPV-16 prototype, while 57% corresponded to variants. Interestingly, the majority (87%) of normal smears had HPV-16 prototype, whereas variants were detected mainly in SIL and ICC. LCR sequencing yielded 80% of variants, including 69% of European, 19% Asian-American, and 12% Asian. We identified a new variant, the Argentine Quechua-51 (AQ-51), similar to B-14 plus two additional changes: G7842-->A and A7837-->C; phylogenetic inference allocated it in the Asian-American branch. The high proportion of European variants may reflect Spanish colonial influence on these native Inca descendants. The predominance of HPV-16 variants in pathologic samples when compared to normal controls could have implications for the natural history of cervical lesions.     

Genome organization and taxonomic position of human papillomavirus type 47 inferred from its DNA sequence

The complete nucleotide sequence of human papillomavirus type 47 (HPV-47) DNA isolated from the lesion of epidermodysplasia verruciformis (EV) was determined. The computer-aided comparison of HPV-47 with other EV-associated viruses using the available sequence data on them revealed that HPV-47 resembles both HPV-5 and HPV-8 as much as HPV-5 and HPV-8 resemble each other, and it led us to regard these three viruses as one cluster and HPV-19 and HPV-25 as another. The conclusion implies that HPV-47 as well as HPV-5 and HPV-8 is associated with the cancer occurrence in EV. Two sets of splicing donor and acceptor sequences in HPV-47, which were previously shown to work in vivo, are also conserved in HPV-5 and HPV-8. One of them allows formation of an ORF predicted to encode an E1/E4 fused protein.