Papilloma virus won’t give up its weapons easily
Human papillomavirus (HPV) is a very common DNA virus that infects the differentiated squamous cell epithelium and mucosal membranes.
Most HPV infections are cleared by the immune system and do not result in clinical complications. However, there are about 100 strains of HPV that affect different body parts, and several of these are known to be oncogenic (by altering epithelial cell replication or disrupting the cell cycle).
It is unclear why only some papillomavirus infections lead to the development of malignant tumours. Also, how can HPVs that share a high level of sequence similarity be so different in terms of tropism and infect different regions of the body? Our understanding of papillomavirus infection has long been hindered by a lack of tissue culture systems for propagating the virus, a lack of animal models for HPVs, and difficulties in finding animal models of natural infection.
Small players with big impact
In their recent research paper, Chirayil et al. have attempted to better characterize the life cycle of these pathogens and their interaction with the host by testing the ability of numerous PV lineages to express microRNAs (miRNAs). miRNAs are small regulatory RNAs of approximately 22 nucleotides that regulate mRNA translation and decay by docking to specific target mRNAs. Members of diverse virus families (herpesvirus, retroviruses, polyomaviruses) that are able to undergo long-term persistent infection encode miRNAs, which seem to play a role in various steps of viral infection and can control immune response, cell death, transformation, and virus gene expression. Notably, these are all DNA viruses (or viruses that have a DNA component to their lifecycle) that have access to the nucleus, where key miRNA processing machinery resides.
Although it is well-documented that HPV can alter the host miRNA repertoire, likely contributing to the biology of cancer, only a few HPVs have been examined, and no widely accepted PV miRNAs had been discovered. This has largely been because of the lack of a good laboratory model for detecting them.
Chirayil et al. have developed a new wet bench technology called microRNA Discovery by forced Genome Expression (miDGE), which can identify miRNAs from genomes for which complete transcriptomes are not readily available. This methodology can screen numerous pathogen genomes in parallel, generating a library of numerous overlapping genomic segments of DNA and subcloning them behind a heterologous RNA polymerase promoter. The logic behind this is that miRNA genes are compact and should be readily expressed by heterologous promoters: the miDGE library is then transfected into mammalian cells, and small RNAs are harvested and sequenced. Next, computational methods are used to screen miRNA transcripts for hallmarks of processing by the miRNA biogenesis machinery (e.g., signatures of bona fide miRNAs, including the presence of a predicted stem-loop structure and clearly defined 5’ ends, as processed by Dicer and DROSHA, and being highly active in RISC). The researchers collected 113 cloned PV genomes from both human and non-human animal sources and screened their genomes for the ability to encode microRNAs.
By this approach, the authors identified only five new highly probable/bona fide miRNAs originating from four different PV genomes: one each from HPV41, HPV17, HPV37, and two from PcPV1. The miDGE technology is well suited to identifying miRNAs from complex multi-genome expression libraries. However, the researchers found that most PVs do not encode miRNAs, including the high-risk cancer types 16, 18, and 45. To further evaluate this, they analyzed the RNA transcriptomes of 42 cervical carcinomas (part of the Cancer Genome Atlas), which presented the entire HPV viral genome: none of these tumours expressed canonical HPV-derived miRNAs. This allowed the researcher to conclude that it is highly unlikely that high-risk HPVs 16, 18, 31, 45, and 58 express canonical viral-encoded miRNAs.
Are these miRNAs expressed in infected tissues?
Together with some experiments to validate the biogenesis and activity of these candidate miRNAs, the researchers went on to verify the detectable expression of these miRNAs in infected tissues. FcPV1 infection is recognized as a cause of macroscopic proliferative skin lesions affecting the legs and feet of chaffinches: a pathology referred to as “leg lesions”. Therefore, they investigated the presence of the candidate PV miRNAs in available leg lesion samples from wild chaffinches. Consistent with the miDGE analysis, they could confirm that PV infection gives rise to miRNAs.
What are the functions of the PV miRNAs?
Previous studies in polyomavirus have demonstrated that polyomavirus miRNAs directly regulate early viral transcripts. To examine the possibility that also PV miRNAs could regulate early viral gene expression, the authors applied bioinformatic analyses and, indeed, found that the FCPV1 and HPV41 miRNAs were able to directly regulate transcripts corresponding to the PV early genomic region.
Involved in terminal differentiation processes, miRNAs have previously been reported to have a significant role in the development of human cancer. Also, microRNA patterns are tissue-specific and their role as post-transcriptional regulators seemed a promising candidate for how PVs interact with the host.
But…if miRNAs are not among the mechanisms for how PVs regulate their and their host’s gene expression, further work will be needed to reveal the weapons of PV’s arsenal.
