Cytopathic effect in human papillomavirus type 1-induced inclusion warts: in vitro analysis of the contribution of two forms of the viral E4 protein.

Myrmecia warts induced by human papillomavirus type 1 (HPV1) are characterized by abundant eosinophilic inclusions associated with HPV1 E4 gene products. The major HPV1 E4 proteins are a 17-kilodalton (kDa) E1-E4 fusion protein and a 16-kDa species lacking the five E1 amino acids and a few E4 residues. To study the contribution of E4 proteins to the formation of myrmecia inclusions, we used a previously designed transient expression system in the rabbit VX2-R keratinocyte line. We find that the E1-E4 and an E4 protein without the E1 residues (E4-3200) form eosinophilic inclusions. Ultrastructural and immunoelectron microscopic studies show that the electron-dense, keratohyalin-like myrmecia inclusions are recognized by anti-E4 antibodies. They are associated with tonofilament bundles at their periphery in the cytoplasm or free of filaments in the nucleus. The E1-E4 inclusions formed in vitro are also homogeneously electron dense, and are usually associated with tonofilaments at their periphery in the cytoplasm and free of filaments in the nucleus. The E4-3200 inclusions are exclusively cytoplasmic and heterogeneously electron dense, with a fibrillar structure made of entangled 10-nm filaments. The expression of either protein in VX2-R cells does not result in the collapse of the cytokeratin network, as shown by immunofluorescence double-labeling experiments. This is in contrast to data reported for the HPV16 E1-E4 protein. Our findings indicate that the E1-E4 protein by itself accounts for the formation of myrmecia inclusions, and suggest that the five N-terminal E1 amino acids play a major role in the interaction of E4 proteins with intermediate filaments.

An arginine-faced amphipathic alpha helix is required for adenovirus type 5 e4orf6 protein function.

A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic alpha helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this alpha helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic alpha helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.