Analysis Of Mutations And Adaptive Evolution Of E2 Genes Of High-risk Human Papillomavirus (HPV) In The Patients With Cervical Cancer In Sichuan Area Of China

bjective:High-risk Human papillomaviruses (HR-HPV) are associated with cervical cancer,which is the second most common cancer in women worldwide. Clinical, molecular, and epidemiological investigations have identified persistent infection with HR-HPV as the major cause of cervical cancer. It is composed of a C-terminal DNA-binding domain, an N-terminal transactivation domain and a flexible hinge. HPV E2 protein plays key roles in the regulation of viral gene transcription and DNA replication.The E2 protein can represse the transcription of the oncogenes E6 and E7. Mutation and integration of the E2 protein is associated with carcinogenesis of HPV. The genome integration of HPV usually disrupts E2 gene and causes an absence of E2 gene sequences. The lack of E2 gene could lead to the upregulated expression of E6 and E7 protein. To study the features of mutations and adaptive evolution of E2 genens of high-risk Human Papillomavirus in patients with cervical cancer in Sichuan area of China.Methods:Histolo-gically confirmed cervical cancer tissues were obtained from 206 patients in Sichuan area between 2008 and 2009. DNA was extracted from frozen tissue specimens by DNeasy Tissue Kit (QIAGEN) and the quality of extracted DNA was checked by PCR amplification of B-globin gene HPVgenotype was detected by a combination of MY09/11 consensus primers PCR and type-specific E6 E7 gene nested PCR The fragments of E2 genes of high-risk HPV including HPV16, HPV18, HPV31, HPV33, HPV45,HPV52,HPV58,and HPV59 were amplified by nested PCR. All positive PCR products of nested PCR were sequenced after they were purified. The mutations were analyzed and determined by Vector NTI 6 software.The HPVs prototype from GenBank was used as reference. Moreover,the phylogenetic trees were constructed by the minimum-evolution and neighbor-joining algorithm using MrBayes v3.1.2 and Mega software version 4.0.To investigate the molecular basis of HPVs evolution, Phylogenetic analysis by maximum likelihood were used to identify lineages and amino acid sites under selective pressure.Finally,we predict the higher structure of the HPV E2 proteins and energy with SWISS-PDBViewer software. Results:In some specimens of all type of HPVs, the complete or part HPVE2 gene failed to be amplified, suggesting that the HPV DNAs in those specimens lost some region of viral E2 gene and exist in the integrated form.The point mutation of the E2 genes of high risk were found in all of samples.Compared with these prototypes published in Genbank,there were several new point mutations in the E2 genes of high-risk HPV in Sichuan area except for HPV31 which had only one. Nucleotide variation within HPV16,18,31,33,45,52,58 and HPV59 was 1.82%,1.28%,0.09%,1.04%,1.26%,1.45%,0.65% and 1.53%, respectively, and amino acid variation was 4.93%,2.19%,0.27%,1.98%,1.63%,2.99%,0.84%,and 3.51%,respectively. Our results showed that10,1,1and58 codon sites in the E2 open reading frames (ORFs) of HPV16,HPV18, HPV33, and HPV59 had nonsynonymous/synonymous substitution rate ratios (dN/dS) over 1,however, no specific codons in E2 ORF of HPV31,HPV45,HPV52 and HPV58were under positive selection.Conclusion:These data indicate that the genome integration of HPV usually disrupts E2 gene and causes an absence of E2 gene sequences.There are also some mutations in the E2 ORFs. Although most sites were under purifying selection or neutral evolution, these data indicative the E2 ORFs of HPV16,HPV18, HPV33, and HPV59 were under positive Darwinian selection. In addition, no position in the E2 genes of HPV31,HPV45,HPV52,and HPV58 isolated from Sichuan area of China was under positive selection. Since these positive sites and mutations were located in different functional sequence segments,it may conclude that these sites are crucial to related function.Especially, the substantial E2 amino acid variation located in surface epitopes could affect efficiency of infection or alter viral immunogenicity. It is reasonable to antieipate that this study would provide potential values to understand the structure and function of HPV E2 proteins and a basis of data and a reference for future studies on cervical carcinogenesis,the evaluation of viruses and immunity.