Rodent Papillomaviruses

Abstract

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.

Keywords: murine papillomavirus; papillomaviruses; preclinical models; rodent papillomaviruses.

Figure 1

Genome organization of rodent papillomaviruses compared to high-risk papillomaviruses. Rodent PVs follow similar genome organization as prototype papillomaviruses and contain early and late open reading frames (ORFs). Shown here is the mouse papillomavirus (MmuPV1) genome (top) as an example of rodent papillomaviruses. For comparison, genomes of prototype high-risk HPVs i.e., HPV5 (bottom left, cutaneous Human Papillomavirus (HPV) as an example of beta-papillomaviruses) and HPV16 (bottom right, mucosal HPV as an example of alpha papillomaviruses) have been shown. The early ORF of MmuPV1 and HPV5 consists of E1 (blue), E2 (pink), E4 (orange), E6 (red) and E7 (purple) and the late ORF consists of L2 (green), L1 (lime). At the genomic level, the most notable change in rodent PV organization compared to HPV16 is the lack of E5 oncogene (brown), which is present in HPV16. Genes E5, E6, E7 have been shown to be oncogenic in the context of several papillomaviruses. The long control region (LCR) is shown in black. E1^E4 (dark brown) and E8^E2 (light brown) splice products are also indicated based on transcript maps of these viruses.

Figure 2

Phylogenetic analysis of rodent PVs. Phylogenetic tree construction of rodent PVs was performed on the basis of L1 nucleotide sequences as per the standard criteria previously set for PV classification [72]. The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model, and the tree with the highest log likelihood (−31,759.0931) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Values less than 50% are not shown. Initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.9374)). The rate variation model allowed for some sites to be evolutionarily invariable (9.1152% sites). The tree is drawn to scale with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 1311 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Version 7.0.21, Pensylvania State University, Hershey, PA, USA) [73]. In this small-scale phylogenetic analysis 33 papillomaviruses that represent prototype papillomaviruses in each clade were chosen for the analysis. For the most recent complete phylogenetic analysis of all papillomavirus species, the reader is referred to de Villiers, (2013) [74].

Figure 3

Progression of MmuPV1-induced tail papillomas in BALB/C-FoxN1^nu nude mice. This figure has been adapted and modified from Xue, et al. (2017, in press) [102]. Nude mice were infected with MmuPV1 virus extract following scarification and tissue was harvested at days 1, 10 and 21 post-infection. Panel A shows H&E staining of infected tissue at indicated time points post-infection with MmuPV1 and the corresponding L1 immunofluorescence (green) to show presence of MmuPV1 capsid proteins. At day 1 we see presence of a scab and immune infiltration but no L1 was detected [102]. At day 10 post-infection we can see hyperplasia, and formation of fibrillary projections begin to appear indicating formation of papilloma. At this time point we can detect L1 in the suprabasal layers. At day 21, we can see more pronounced papillomatosis, and L1 staining can be seen even in basal layers of the papilloma. The H&E images and fluorescent images were captured using a Zeiss AxioImager M2 microscope and AxioVision software version 4.8.2 (ZEISS, Jena, Germany). Panel B shows a high resolution wide-field image of an MmuPV1-induced tail wart at 6 months post-infection. In this image we have shown L1-K14 dual immunofluorescence. Extensive papilloma formation coupled with expansion of the K14 layer (green) is seen, and L1 expression (red) is seen throughout the epithelia (inset). The nuclei are stained with DAPI (blue). High resolution wide-field fluorescent images were acquired by means of a super-resolution Leica SP8 STED confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA) equipped with a motorized stage located in the UW optical core. This microscope is equipped with PMT and HyD lasers. All of the images were taken by means of a 20× objective lens (Specifications: HC PL APO 20×/0.75 CS2, Dry). The images were acquired by tile-scanning by marking positions around the region of interest on the LAS-X suite (version: 2.0.1). The merged wide-field image was obtained by automatic stitching of individual styles by means of an in-built auto-stitching algorithm that is part of the LAS-X suite. Scale bar (100 μm) is shown in top right corner.

Figure 4

Transcription map of MnPV1. This figure has been adapted and modified from [97]. The full transcription map for MnPV in productive lesions was assembled from PCR, RACE, and RNA-seq data. At the top, the genome organization of MnPV is presented for better understanding; numbers indicate the position of ORF starts and ends. Mapped TSSs 78 and 710 and pA cleavage sites 4370-6 and 7317 are depicted. For transcripts, exons are represented by black boxes (when coding for a particular ORF) while introns are marked as thin solid lines. Positions of the splice junctions in each transcript are given by numbers at the top.

Figure 5

Transcription map of MmuPV1. This figure has been modified and adapted from Xue, et al. (2017) (Manuscript accepted [102]) The bracket line in the middle of the panel represents a linear form of the virus genome for better presentation of head-to-tail junction, promoters (arrows), and polyadenylation cleavage sites, early pA CS 3864 and late pA CS7063. The open reading frames (ORFs) are diagramed above the bracket line as colored boxes, and the numbers above each ORF are nucleotide positions of the first nucleotide of the start codon and the last nucleotide of the stop codons in the MmuPV1 genome. LCR indicates a long control region. Below the bracket line are the RNA species derived from alternative promoter usage and alternative RNA splicing. Exons (black boxes) and introns (thin lines) are illustrated for each species of the RNA, with the mapped splice site positions numbered by nucleotide position in the virus genome and coding potentials are shown on the left.