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The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an unexpected difficulty experienced when trying to clone HPV33 long control region (LCR) variants in  Escherichia 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 in  E. 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 species  Alphapapillomavirus 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.   Electronic supplementary material  The online version of this article (10.1007/s11262-020-01754-4) contains supplementary material, which is available to authorised users.

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