The abundant human papillomavirus type 11 (HPV 11) E1∧E4,E5 transcript potentially encodes the E1∧E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1∧E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1∧E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3′ end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1∧E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1∧E4,E5a construct that lacked the 5′ noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1∧E4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1∧E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1∧E4,E5 transcript is a functional message that can support both E1∧E4 and E5a expression in eukaryotic cells.
Neutralization of virus is likely to be necessary for development of an effective prophylactic vaccine against genital human papillomavirus (HPV) infection. Two New Zealand white rabbits were immunized with purified HPV type 11 (HPV 11) virions in Freund's adjuvant. An enzyme linked immunoassay (ELISA) was used to determine the quantity of IgG which recognized the HPV 11 major capsid protein (L1 protein) virus-like particles (VLPs) in the two anti-HPV 11 sera (serum A and serum B). The concentration of HPV 11 L1 VLP-specific IgG in the A and B sera were determined to be 37 and 90 micrograms per ml, respectively. The A and B sera were used in neutralization experiments in the athymic mouse xenograft system with known quantities of purified HPV 11 virions. The concentration of HPV 11 L1 VLP-specific IgG required to neutralize HPV 11 was determined for each antiserum. This concentration of IgG was approximately 700 to 900 ng per ml. This study demonstrates a positive correlation between the level of HPV 11 L1 VLP-specific IgG in animals immunized with HPV 11 virions and neutralization of HPV 11 in the athymic mouse model. Further studies are needed 1) to determine if sera or genital secretions from other species are neutralizing in the athymic mouse xenograft system, and 2) to determine if the VLP ELISA can be used as a reliable substitute for more cumbersome neutralization assays.
During normal keratinocyte differentiation, a coordinated expression of many cytoskeletal and regulatory proteins occurs. Several studies suggest that expression of some of these proteins is altered in epithelium infected by the human papillomavirus (HPV). To examine protein expression, human foreskin tissue was infected with either the low-risk HPV type 11 or with HPV 83, a high-risk type. The foreskin tissue was implanted and grown in the athymic mouse xenograft system. Immunohistochemistry and immunoblot analysis of human foreskin xenografts were performed to detect cytokeratin 16 (K16), a protein previously identified in proliferative disorders of the skin. K16 was abundant in HPV 11-infected xenograft tissue, but was not detected in uninfected or HPV 83-infected tissue. Analysis of protein extracted from human biopsy tissue demonstrated the same expression patterns in natural infection by HPV 11. Reverse transcriptase PCR detected mRNA transcripts for K16 in both experimental and natural HPV 11-infected tissues, but not in uninfected tissue. These studies suggest that K16 overexpression during HPV 11-infection is regulated at the level of transcription. The marked epithelial proliferation that occurs in HPV 11 infection may involve alterations in expression of cytoskeletal proteins, including K16. Determining the mechanisms of K16 transcriptional induction could lead to therapies with the ability to reduce cell proliferation within infected tissue.
