Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [ 35 S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The ␣ 6 ␤ 4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the ␣ 6 or ␤ 4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An ␣ 6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-␤ 4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the ␣ 6 ␤ 4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of ␣ 6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the ␣ 6 integrin subunit and that integrin complexes containing ␣ 6 integrin complexed with either ␤ 1 or ␤ 4 integrins may act as a receptor for PV binding and entry into epithelial cells.

Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the alpha6 integrin (Evander et al., J. Virol. 71, 2449-2456, 1997). We have further investigated the role the alpha6 integrin plays in PV binding. Here we show that the cells expressing the alpha6 integrin, partnered with either the beta4 integrin or the beta1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the alpha6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human alpha6A gene. The transduction of the alpha6 integrin gene into DG75 cells results in the cell surface expression of the alpha6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for alpha6 integrin. Furthermore, the alpha6 protein is partnered with the beta1 integrin in DG75 cells. Finally, we show that the DG75-alpha6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-alpha6 antibody. Together these data indicate that the alpha6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding.

It is now well recognized that cervical cancer is caused by infection with certain human papillomavirus (HPV) subtypes and while interferon-alpha (IFN-alpha) is used to treat HPV-infected lesions, HPV appears to have developed a means to avoid the effects of IFN-alpha. Clinically, resistance appears to be associated with the expression of the E7 oncoprotein. Here we investigated the effects of expression in cells of the E7 protein from high- and low-risk papillomavirus subtypes on a range of responses to IFN-alpha. 2fTGH, a cell line dependent on IFN-alpha for growth in selection medium, grew significantly less well in the presence of E7, and the antiproliferative effects of IFN-alpha upon epithelial cells was lost upon E7 expression. The antiviral effects of IFN-alpha were abrogated in E7-expressing cells. Loss of response to IFN-alpha was found to occur in both high- and low-risk papillomaviruses. Finally, deletion of amino acids 21-24 of HPV type 16 E7 protein partially reversed repression. We conclude that E7 inhibits the functional effects of IFN-alpha and that this property is shared by all HPV subtypes tested.

There are a number of observations that suggest the dsRNA-activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full-length PKR is present but not active in 21 of 28 patient samples from B-cell chronic lymphocytic leukemia (B-CLL). PKR from these patients was unable to auto-activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58 IPK . We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B-CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL.

Identification of appropriate reference genes (RGs) is critical to accurate data interpretation in quantitative real-time PCR (qPCR) experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq) to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.