Identification of six putative novel human papillomaviruses (HPV) and characterization of candidate HPV type 87

Abstract

Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that the candHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.

FIG. 1

Phylogenetic trees of Supergroup A (mucosal) HPVs. (a) Partial L1 region encompassed by primers MY11/09, with group division at ancestor branching. The sequences described in this work are printed in boldface. (b) Complete genomes of all available HPV sequences, with group divisions.

FIG. 1

Phylogenetic trees of Supergroup A (mucosal) HPVs. (a) Partial L1 region encompassed by primers MY11/09, with group division at ancestor branching. The sequences described in this work are printed in boldface. (b) Complete genomes of all available HPV sequences, with group divisions.

FIG. 2

Genome organization and PCR clones of candHPV87. ORFs are represented as thick arrows, and numbers show nucleotide positions of start and stop codons; clones are represented as thin arrows. Clones L1-E1 16 and L1-E1 37 were derived from the E1-L1 amplified product, E1-L1 11/2 and L1atg were derived from the E1 L1 amplified product; clone E11-E2as was subsequently derived from an independent PCR product in order to complete the E1-E4 region and clone MY 16 was derived from a MY11-MY09 PCR product in order to complete the genome in the L1 gene.

FIG. 3

Growth enhancement properties of the E6 and E7 proteins of HPVs in transient transfection experiments and foci formation. (a) Growth of HaCat keratinocytic cells 4 days after transfection by using HFV-based vectors (values from three independent experiments, labeled 1, 2, and 3). (b) Growth of NIH 3T3 mouse fibroblasts 4 days after transfection by using HFV-based vectors (values from three independent experiments, labeled 1, 2, and 3). (c) Focus formation in NIH 3T3 fibroblasts (in foci per well). lipof., negative control in which no plasmid was transfected but cells were treated with Lipofectin.