Amplification and sequencing of a 190bp segment containing nucleotide 6240 from prototype human papillomavirus16 L1 gene and 10 clinical isolates from the UK

Abstract number: 1733_938

Al Zoubi H., Vallely P., Turner A., Guiver M.

Previous studies have shown that all HPV16 clinical isolates, for which sequence data is available, have a cytosine to guanosine (C to G) nucleotide change at position 6240 relative to the prototype HPV16 sequence. The coordinate L1 amino acid 202 resulting from this change is aspartic acid instead of histidine (D202H). This change has been suggested to be important to escape the immune response and was also found to be important for the efficient assembly of HPV16L1 recombinant proteins into virus-like particles which are currently being used as a vaccine against HPV infections. An HPV16 genome cloned into pAT153 plasmid was obtained by our laboratory in 1986. However, no information on the source of the cloned isolate was available. In an attempt to identify the cloned HPV16 isolate for further L1 proteins expression study, we amplified and sequenced a 190bp segment of L1 gene which contained the coding region for the nucleotide of interest (6240). In this isolate nucleotide 6240 was found to be C and the correspondent amino acid at codon 202 in which the C nucleotide is found was therefore histidine. Based on the previously published data and on the NCBI BLAST alignment, we identified the cloned HPV16 to be the prototype HPV16 isolate. To find an appropriate HPV16 isolate for protein expression and to confirm the presence of the D202H observation in clinical isolates, 10 clinical isolates collected from UK patients with different degrees of cytological abnormality were also amplified and sequenced using the same PCR and sequencing conditions. All clinical isolates were found to contain nucleotide G at position 6240 coding for aspartic acid at position 202 in the L1 gene.

The established PCR and sequencing conditions could be used to confirm the presence of D202H in HPV16 isolates before proceeding to protein expression of HPV16L1. It could also be used to sequence larger numbers of clinical isolates from patients with different degrees of cytological abnormality, which will further help in understanding the HPV16 associated pathology.