Sequence divergence yet conserved physical characteristics among the E4 proteins of cutaneous human papillomaviruses.

Human papillomavirus (HPV) types 1, 2, and 4 together comprise the major cause of cutaneous papillomas in the general population. We have aligned the genomes of these three viruses by partial sequence analysis, and have sequenced the E4 open reading frames (ORFs) of HPV 2 and HPV 4. After expression as beta-gal fusion proteins in bacteria, antibodies raised to the putative E4 gene-products of both virus types were used to identify the native E4 proteins in naturally occurring tumors. At the primary amino acid sequence level, the E4 protein of HPV 2 was found to be most homologous with those of HPV 6 and 11 and was not closely related to those of HPV 1 or 4. Although the E4 ORF represents a region of weak homology amongst papillomaviruses, the E4 encoded proteins showed significant conservation in their physical characteristics. Like those of HPV 1, the E4 proteins of both HPV 2 and HPV 4 were found to be composed of a major low-molecular-weight doublet (16.5/18K for HPV 2, 20/21K for HPV 4, c.f. 16/17K for HPV 1) along with minor high-molecular-weight species, which probably represent dimers of the smaller proteins, (33K for HPV 2, 40K for HPV 4, c.f. 32/34K for HPV 1). The E4 products of all three virus types were multiply charged, and exhibited a characteristic migration pattern following alkaline urea gel electrophoresis. Although the levels of E4 expression in tumors induced by the different virus types was very different, this was found to correlate closely with the level of virus production characteristic of each virus type. In all three cases, E4 proteins were found to be primarily cytoplasmic, and to be associated with the distinctive cytoplasmic inclusion granules characteristic of each virus type. The poor sequence conservation between the E4 protein of HPVs 1, 2, and 4, taken alongside the ability of these viruses to infect similar histological sites, suggests that E4 may not be involved in determining tissue specificity. Our results suggest conserved physical characteristics (acidic, multiply charged, ability to form dimers) and similar site of expression may be the important factors for E4 function.

Phosphorylation of the human papillomavirus type 1 E4 proteins in vivo and in vitro.

Following incubation of HPV 1-induced warts in the presence of [32P] phosphate several of the E4-encoded proteins were found to be radiolabeled. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 17K E4 polypeptides had incorporated [32P]phosphate whereas those of 16K were unlabeled. Purified E4 gene products were separated by ion exchange chromatography into a large number of different species, which were of similar size but of different charge due to varying extents of phosphorylated peptides have been isolated and identified. Phosphoserine and phosphothreonine were identified in all 16/17K E4 fractions but not phosphotyrosine. Both HPV 1 E4 16K and 17K fractions were phosphorylated in vitro by cAMP-dependent protein kinase but not by myosin light chain kinase or by phosphorylase kinase. Incubation with cAMP PK gave incorporation of approx. 0.5 mole phosphate/mol of protein indicating that the cAMP-dependent protein kinase site(s) was partially phosphorylated in vivo. This view was supported by the fact that species which were more heavily phosphorylated in vivo incorporated less phosphate after cAMP-dependent protein kinase phosphorylation. HPV 1 E4 was also phosphorylated at serine and threonine residues by a crude cytoplasmic extract prepared from cultured human keratinocytes and cultured human retinoblasts. These results are discussed in the light of the known effects of phosphorylation on the interactions of other keratinocyte-specific proteins.

Analysis of HPV-1 E4 gene expression using epitope-defined antibodies.

Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV-1. Five of these were found to recognize denaturation-resistant epitopes as determined by Western blotting--and their binding sites were identified by determining their reactivity against a panel of bacterial E4--beta-galactosidase fusion proteins which contained progressive deletions at the C-terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope-defined mAbs, along with anti-peptide antibodies raised against putative N- and C-terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV-1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5' end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N-terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS)