RNA sequencing analysis identifies novel spliced transcripts but does not indicate quantitative or qualitative changes of viral transcripts during progression of cottontail rabbit papillomavirus-induced tumours.

Persistent infections with high-risk human papillomaviruses (HPVs) can result in the development of cancer of the cervix uteri and other malignancies. The underlying molecular mechanisms leading to the progression of HPV-induced lesions are, however, not well understood. Cottontail rabbit papillomavirus (CRPV) induces papillomas in domestic rabbits which progress at a very high rate to cancer. Using this model, we compared the transcriptional patterns of CRPV in papillomas and carcinomas by RNA sequencing (RNA-seq). The most abundant transcripts can encode E7, short E6 and E1^E4, followed by full-length E6, E2, E1 and E9^E2C. In addition, we identified two rare, novel splice junctions 7810/3714 and 1751/3065 in both papillomas and carcinomas which have been described for other papillomaviruses. Neither RNA-seq nor quantitative real-time PCR-based assays identified qualitative or quantitative changes of viral transcription between papillomas and carcinomas. In summary, our analyses confirmed that papillomaviruses have highly similar transcriptional patterns, but they do not suggest that changes in these patterns contribute to the progression of CRPV-induced tumours.

A novel recombinant papillomavirus genome enabling in vivo RNA interference reveals that YB-1, which interacts with the viral regulatory protein E2, is required for CRPV-induced tumor formation in vivo.

YB-1 is considered a negative prognostic marker for different types of cancer. Increased YB-1 protein levels in tumor cells indicate a worse prognosis. In a preceding study comparing the transcripts of CRPV-induced benign papillomas to mRNA levels of malignant epithelial tumors, we identified YB-1 as a gene that is up-regulated in papillomavirus-associated carcinomas and which causes an invasive phenotype in CRPV-positive cells in vitro. Here we demonstrate that YB-1 is a previously unknown factor required for papillomavirus-induced tumor development in the rabbit animal model system. By infecting the animals with a novel recombinant shRNA-expressing CRPV genome, we show that knock-down of YB-1 dramatically reduces papillomavirus-dependent tumor formation in vivo. Consistent with previous reports showing a nuclear distribution of YB-1 proteins as a hallmark of malignancy, we demonstrate a predominantly nuclear localization of YB-1 in CRPV-immortalized cells. Furthermore we give evidence of YB-1 regulating the CRPV URR and thereby viral gene expression and we identified YB-1 as a novel interactor of the CRPV regulatory protein E2. Taken together we hypothesize that YB-1 is essential for papillomavirus-induced tumor formation probably by regulating viral gene expression including expression of the oncogenes E6 and E7.

A recombinant cottontail rabbit papillomavirus genome for ectopic expression of genes in cells infected with virus in vivo.

The objective of this study was to construct a cottontail rabbit papillomavirus (CRPV) genome that would co-express a gene of choice and the viral genome simultaneously. Using this construct, the effects of the ectopic expression of diverse viral or cellular genes on PV-infected cells can be examined to elucidate which genes are essential for tumor formation. CRPV-pLAIIdelXba1, which lacks the major portion of L2 (designated the XbaI fragment), has been previously shown to fully retain the ability to induce tumors, and this ability was confirmed in this study. Insertion of the XbaI fragment in an antisense orientation did not change the efficiency of tumor induction. An SV40 overexpression cassette that originated from pSG5 and contains a more diverse multiple cloning site (MCS) was cloned into CRPV-Xba1-mcs, a CRPV genome based on CRPV-pLAIIdelXba1 that contains an additional MCS inserted via XbaI digestion. Additionally, the L1 ATG initiation codon of this construct, designated CRPV-Xba1-oe-WT, was mutated to avoid unnecessary L1 protein expression, which produced the CRPV-Xba1-oe-L1mut construct. Injection of these constructs into two New Zealand White rabbits and monitoring of tumor growth for two to six months showed that CRPV-Xba1-oe-WT induced tumors at 1/10 and 1/10 of the injection sites in two animals, while the control injections in each rabbit induced tumors at 3/10 and 4/10 injection sites, respectively. However, CRPV-Xba1-oe-L1mut induced tumors at 3/10, 6/10, 7/12 and 11/12 sites in four injected animals, and the control injections induced tumor growth in these animals at 6/10, 10/10, 12/12 and 12/12 of the injected sites, respectively. Thus, CRPV-Xba1-oe-L1mut could potentially be used to conduct overexpression experiments in vivo that can be used to measure the negative or positive influences of ectopically expressed foreign or HPV genes on tumor growth.