Role of Conserved Cysteine’s and Methionine’s in Human Papillomavirus-16 Major Capsid Protein L1

Abstract

Human Papillomavirus (HPV) is tumor inducing virus which infects mucosa and cutaneous keratinocytes. HPV is the causative agent for cervical cancer. The cervical cancer induced death rate is high among women in developing countries. There are various cancer causing subtypes of HPVs. The first aim of the current study was to find the most prevalent HPV subtype among the local cervical cancer patients. For that purpose, samples were collected from the patients of different ethnic and social back grounds (mostly from the twin cities Islamabad & Rawalpindi) visiting a Government tertiary care hospital between 2010-2012. A total of 67 formalin fixed paraffin-embedded sections of cervical lesions were obtained. PCR-based detection method showed the presence of HPV DNA in 59 samples. HPV16 was present in 30 samples, while 22 samples were positive for HPV18. A small number of HPV positive samples (7) were found to be HPV16 and HPV18 negative, representing a minor percentage (10%). However HPV16 and HPV18 accounted for 44.8% and 32.8% of samples respectively. In the absence of therapy for an established HPV infection, there is a huge reliance on prophylaxis. The available prophylactic vaccines are mainly based on the self-assembly of the major capsid protein L1 into a virion-like icosahedral particle. After disruption of the intermolecular disulphide bonds, each icosahedral particle, dissociates into 72 pentameric capsomeres. Earlier, it has been proposed that C161, C229, and C379 may be necessary for the integrity of L1-capsids probably through intramolecular bonding. Therefore, the next objective of the current study was to explore the potential role of C161, C229, and C379 residues in HPV16 genome assembly, encapsidation and infectivity using organotypic tissue culture. Abstract xvi In order to do so cysteine residues; C161, C229, and C379 were mutated to serine through site directed mutagenesis. Linearized DNA from wild type and mutant HPV16 genome was electroporated in Human forskin keratinocytes (HFK’s) and respective organotypic raft cultures were established. The comparison of crude viral preps from mutant and wild type HPV16 showed that endonuclease-resistant genomes in mutant viral preps was lower than that of wild type. It may be concluded that C161S, C229S and C379S are critical for proper formation of endonuclease resistant capsids. Furthermore, these mutants were found to cause 50% or more reduction in viral infectivity. To summarize, this study contribute to the current understanding about the conserved cysteine residues; C161, C229, and C379 and upstream methionine residues present in major capsid protein L1 in HPV16, maturation and more importantly in viral infectivity, also showing importance of using organotypic raft culture for studies. Next objective was to determine the role of two upstream methionine residues in the biology of HPV16L1, within the natural environment of Human epithelial tissue. In order to do so, HFK’s were electroporated with HPV16L1 methionine mutant 1(Met 1 with translational initiation at first methionine) and HPV16L1 methionine mutant 2 (Met 2 with translational initiation at second methionine). The infectivity assay showed higher infectivity in Met 1 as compared to Met 2.