Abstract
MicroRNAs (miRNAs) are small RNAs that regulate diverse biological processes including multiple aspects of the host-pathogen interface. Consequently, miRNAs are commonly encoded by viruses that undergo long-term persistent infection. Papillomaviruses (PVs) are capable of undergoing persistent infection, but as yet, no widely-accepted PV-encoded miRNAs have been described. The incomplete understanding of PV-encoded miRNAs is due in part to lack of tractable laboratory models for most PV types. To overcome this, we have developed miRNA Discovery by forced Genome Expression (miDGE), a new wet bench approach to miRNA identification that screens numerous pathogen genomes in parallel. Using miDGE, we screened over 73 different PV genomes for the ability to code for miRNAs. Our results show that most PVs are unlikely to code for miRNAs and we conclusively demonstrate a lack of PV miRNA expression in cancers associated with infections of several high risk HPVs. However, we identified five different high-confidence or highly probable miRNAs encoded by four different PVs (Human PVs 17, 37, 41 and a Fringilla coelebs PV (FcPV1)). Extensive in vitro assays confirm the validity of these miRNAs in cell culture and two FcPV1 miRNAs are further confirmed to be expressed in vivo in a natural host. We show that miRNAs from two PVs (HPV41 & FcPV1) are able to regulate viral transcripts corresponding to the early region of the PV genome. Combined, these findings identify the first canonical PV miRNAs and support that miRNAs of either host or viral origin are important regulators of the PV life cycle.
Highlights
Papillomaviruses (PVs) comprise a large family of circular double-stranded DNA viruses
To identify miRNA genes in situations where transcripts are not obtainable, we developed the approach of miRNA Discovery by forced Genomic Expression. miRNA Discovery by forced Genome Expression (miDGE) relies on generating a library of numerous overlapping genomic segments of DNA from a particular organism or locus and subcloning them behind a heterologous RNA polymerase (RNP) II promoter (Fig 1)
The concept relies on the principle that miRNA genes are compact and should be readily expressed by heterologous upstream RNP II promoters, or in the rare cases that a primary miRNA transcript is driven by RNP III, that these promoters are small and proximal to the miRNA gene so as to be included in miDGE library constructs
Summary
Papillomaviruses (PVs) comprise a large family of circular double-stranded DNA viruses. A minority of these are known as carcinogenic agents [1,2,3], only a small fraction of hosts infected with these high risk types will go on to develop high grade lesions. It remains incompletely understood what factors dictate whether or not HPV infection will develop into malignant cancer [1,3]. Developing a better understanding of PV gene products and their regulation throughout diverse PV lineages provides an evolutionary foundation for deciphering the mechanisms resulting in differential outcomes of infection
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