Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells

In this study, we report an unexpected difficulty experienced when trying to clone HPV33 long control region (LCR) variants inEscherichia coli. Standard cloning strategies proved to be inappropriate to clone HPV33 LCR variants in the forward orientation into a eukaryotic reporter vector (pGL2-Basic). However, by slight modification of culture conditions (incubation at 25  °C instead of 37 °C), constructs containing the HPV33 LCR variants in the forward orientation were obtained. Transformation experiments performed with different HPV33 LCR constructs indicated that there is a sequence element in the 5′ LCR of HPV33 causing temperature-dependent toxic effect inE. coli. Sequence analysis revealed the presence of an open reading frame (ORF) in the 5 ′ part of HPV33 LCR potentially encoding a 116-amino acid polypeptide. Protein structure prediction suggested that this putative protein might have a structural similarity to transmembrane proteins. Even a low-level expression of this protein may cause significant toxicity in the host bacteria. In silico analysis of the LCR of HPV33 and some other HPV types belonging to the speciesAlphapapillomavirus 9 (HPV31, 35 and 58) seemed to support the assumption that the ORFs found in the 5 ′ LCR of these HPVs are protein-coding sequences. Further studies should be performed to prove that these putative proteins are really expressed in the infected host cells and to identify their function.
Source: Virus Genes - Category: Genetics & Stem Cells Source Type: research