Characterization of a Conserved Cysteine Residue in the Papillomavirus E2 Protein

Background: Human papillomaviruses (HPVs) are DNA tumor viruses that infect cutaneous and mucosal epithelium. While most infections are self-limiting, a small subset that infects the mucosal epithelium progresses to cancer. All papillomaviruses encode the protein E2 which regulates viral transcription and replication; a highly conserved cysteine residue in the DNA contact helix of E2 plays an unknown role. Previous research suggests the residue is not necessary for replication or binding to DNA. We hypothesize that post-translational modification of this conserved cysteine residue leads to release of viral DNA during packaging of progeny virions. Methods: Mutations of the murine papillomavirus conserved E2 C307 residue to serine and phenylalanine were used to investigate its role in E2 function. C33A, HPV negative cervical cancer cells, were transfected with an E2-responsive luciferase reporter and either wild type or mutant C307 E2 vectors; luciferase assays were performed 48 hours post-transfection to assess transcriptional activity. Whole cell lysates from overexpressed C307 mutants were separated by SDS-PAGE and immunoblotted to assess expression levels relative to wild type. To examine protein localization, C33A cells were transfected with equal amounts of wild type or mutant E2 and fixed 48 hours post-transfection for immunofluorescence. Results: C307S and C307F mutants are both capable of weakly activating transcription. Overexpression of the mutants resulted in a dose dependent increase in transcriptional activity. Both mutants are expressed at levels comparable to wild type E2 and are correctly localized to the nucleus. Conclusion/Impact: The deficient transcription function displayed by the C307 mutants cannot be explained by poor expression or mislocalization. Continued study of this conserved cysteine will help to further understanding of papillomavirus biology and may offer insight into novel avenues for treatment or prevention of HPV-associated cancers

Characterization and Whole Genome Analysis of Human Papillomavirus Type 16 E1-1374^63nt Variants

Background. The variation of the most common Human papillomavirus (HPV) type found in cervical cancer, the HPV16, has been extensively investigated in almost all viral genes. The E1 gene variation, however, has been rarely studied. The main objective of the present investigation was to analyze the variability of the E6 and E1 genes, focusing on the recently identified E1-1374^63nt variant. Methodology/Principal Findings. Variation within the E6 of 786 HPV16 positive cervical samples was analyzed using high-resolution melting, while the E1-1374^63nt duplication was assayed by PCR. Both techniques were supplemented with sequencing. The E1-1374^63nt duplication was linked with the E-G350 and the E-C109/G350 variants. In comparison to the referent HPV16, the E1-1374^63nt E-G350 variant was significantly associated with lower grade cervical lesions (p=0.029), while the E1-1374^63nt E-C109/G350 variant was equally distributed between high and low grade lesions. The E1-1374^63nt variants were phylogenetically closest to E-G350 variant lineage (A2 sub-lineage based on full genome classification). The major differences between E1-1374^63nt variants were within the LCR and the E6 region. On the other hand, changes within the E1 region were the major differences from the A2 sub-lineage, which has been historically but inconclusively associated with high grade cervical disease. Thus, the shared variations cannot explain the particular association of the E1-1374^63nt variant with lower grade cervical lesions. Conclusions/Significance. The E1 region has been thus far considered to be well conserved among all HPVs and therefore uninteresting for variability studies. However, this study shows that the variations within the E1 region could possibly affect cervical disease, since the E1-1374^63nt E-G350 variant is significantly associated with lower grade cervical lesions, in comparison to the A1 and A2 sub-lineage variants. Furthermore, it appears that the silent variation 109T>C of the E-C109/G350 variant might have a significant role in the viral life cycle and warrants further study

Three novel canine papillomaviruses support taxonomic clade formation

More than 100 human papillomaviruses (HPVs) have been identified and had their whole genomes sequenced. Most of these HPVs can be classified into three distinct genera, the alpha-, beta- and gamma-papillomaviruses (PVs). Of note, only one or a small number of PVs have been identified for each individual animal species. However, four canine PVs (CPVs) (COPV, CPV2, CPV3 and CPV4) have been described and their entire genomic sequences have been published. Based on their sequence similarities, they belong to three distinct clades. In the present study, circular viral DNA was amplified from three dogs showing signs of pigmented plaques, endophytic papilloma or in situ squamous cell carcinoma. Analysis of the DNA sequences suggested that these are three novel viruses (CPV5, CPV6 and CPV7) whose genomes comprise all the conserved sequence elements of known PVs. The genomes of these seven CPVs were compared in order properly classify them. Interestingly, phylogenetic analyses, as well as pairwise sequence alignments of the putative amino acid sequences, revealed that CPV5 grouped well with CPV3 and CPV4, whereas CPV7 grouped with CPV2 but neither group fitted with other classified PVs. However, CPV6 grouped with COPV, a lambda-PV. Based on this evidence, allocation of CPVs into three distinct clades could therefore be supported. Thus, similar to HPVs, it might be that the known and currently unknown CPVs are related and form just a few clades or genera

Genetic Analysis of the Bovine Papillomavirus E2 Transcriptional Activation Domain

AbstractThe bovine papillomavirus type 1 E2 transactivator has a large amino-terminal 215-residue transcriptional activation domain (TAD) that is active inSaccharomyces cerevisiaeand higher eukaryotic cells. Comparison to other transcriptional activators suggests that its functions may be mediated in part through two acidic regions, A1 and A2, in this domain. We have characterized the functional elements within the E2 TAD using LexA–E2 fusions and by screening randomly generated libraries of E2 mutations for transcriptional activation in yeast. The A1 region was highly sensitive to substitutions that reduce negative charge, although there was not a perfect correlation between overall charge and transcriptional activity. Mutations were isolated within a hydrophobic amino acid motif that overlaps the A2 region and resembles elements described in other viral and cellular transactivation domains. When fused to the LexA DNA binding domain, this hydrophobic motif within the acidic A2 region was unable to activate transcription inS. cerevisiae.Multiple highly defective mutations primarily altering hydrophobic amino acids were identified in the distal third of the E2 TAD. The transcription phenotype of many of these E2 TAD mutations was similar in yeast and COS cells

Mutational analysis of human papillomavirus type 16 E6 demonstrates that p53 degradation is necessary for immortalization of mammary epithelial cells

We have previously demonstrated that normal human mammary epithelial cells (MECs) are efficiently immortalized by human papillomavirus type 16 (HPV16) E6. HPV16 E6 binds to and induces p53 degradation in vitro and induces a marked reduction of p53 protein in MECs. Low-risk HPV6 E6 is defective for p53 binding and degradation in vitro but immortalized MECs at low efficiency. The HPV6 E6-immortalized MECs had markedly reduced levels of p53. To directly investigate whether the ability of HPV16 E6 to stimulate p53 degradation is required for E6-induced immortalization, a series of HPV16 E6 mutants were analyzed for the ability to bind and degrade p53 in vitro, induce a reduction in p53 levels in vivo, and immortalize MECs. We observed that one set of mutants efficiently immortalized MECs, caused a reduction in p53 levels in vivo, and degraded p53 in vitro. Other mutants immortalized MECs with low efficiency and either induced p53 degradation at low levels or were unable to induce p53 degradation in vitro; however, all of the immortal clones displayed low levels of p53. A third class of mutants did not immortalize MECs and failed to induce a reduction in p53 levels in vivo or degrade p53 in vitro. These results demonstrate that a reduction in p53 protein levels due to enhanced degradation is essential for MEC immortalization by HPV16 E6.</jats:p

Trans activation by the bovine papillomavirus E2 protein in Saccharomyces cerevisiae

The papillomavirus E2 protein functions as an enhancer-binding factor to promote transcription in mammalian cells. We found that one copy of the E2 binding site acted as an E2 protein-dependent upstream activating sequence in Saccharomyces cerevisiae. Additional copies of the binding motif further augmented transcription. These results imply that the E2 protein functionally interacts with highly conserved transcriptional elements.</jats:p