Analysis of Regulatory Motifs Within HPV Transcripts

Papillomavirus late gene expression is highly dependent on host epithelial cell differentiation, such that capsid proteins are produced only in differentiating cells. Several papillomaviruses contain negative regulatory elements (NREs), that is, short regions of late transcripts that interact with host cellular RNA processing factors to prevent capsid protein synthesis in undifferentiated cells. In this chapter, the human papillomavirus (HPV)-16 NRE will be used as an example to show how cis -acting RNA regulatory elements can be identified and mapped using transient transfection of reporter gene constructs. The use of reporter gene assays is also readily applicable to the identification and characterization of novel promoters and other regulatory sequences in HPV DNA. In vitro RNA-protein binding techniques, including ultraviolet crosslinking, electrophoretic mobility shift assay, and affinity purification of RNA binding proteins, will also be described, again using the HPV-16 NRE as an example. These techniques may be used to identify cellular proteins that bind the NRE, allowing its mode of action to be deduced. They may also be used to study interactions between host cellular proteins and other protein-binding motifs on HPV mRNA. These interactions are important for the regulation of HPV gene expression, and have key roles in splicing, polyadenylation, mRNA export, stability, and translation.