Bovine Delta Papillomavirus E5 Oncoprotein Interacts With TRIM25 and Hampers Antiviral Innate Immune Response Mediated by RIG-I-Like Receptors

Persistent infection and tumourigenesis by papillomaviruses (PVs) require viral manipulation of various of cellular processes, including those involved in innate immune responses. Herein, we showed that bovine PV (BPV) E5 oncoprotein interacts with a tripartite motif-containing 25 (TRIM25) but not with Riplet in spontaneous BPV infection of urothelial cells of cattle. Statistically significant reduced protein levels of TRIM25, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) were detected by Western blot analysis. Real-time quantitative PCR revealed marked transcriptional downregulation of RIG-I and MDA5 in E5-expressing cells compared with healthy urothelial cells. Mitochondrial antiviral signalling (MAVS) protein expression did not vary significantly between diseased and healthy cells. Co-immunoprecipitation studies showed that MAVS interacted with a protein network composed of Sec13, which is a positive regulator of MAVS-mediated RLR antiviral signalling, phosphorylated TANK binding kinase 1 (TBK1), and phosphorylated interferon regulatory factor 3 (IRF3). Immunoblotting revealed significantly low expression levels of Sec13 in BPV-infected cells. Low levels of Sec13 resulted in a weaker host antiviral immune response, as it attenuates MAVS-mediated IRF3 activation. Furthermore, western blot analysis revealed significantly reduced expression levels of pTBK1, which plays an essential role in the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. Our results suggested that the innate immune signalling pathway mediated by RIG-I-like receptors (RLRs) was impaired in cells infected with BPVs. Therefore, an effective immune response is not elicited against these viruses, which facilitates persistent viral infection.

Prohibitin 2 is Involved in Parkin-Mediated Mitophagy in Urothelial Cells of Cattle Infected with Bovine Papillomavirus

Prohibitin 2 (PHB2), an inner mitochondrial membrane (IMM) protein, has recently been identified as a novel receptor involved in parkin-mediated mitophagy. In the field of veterinary medicine, the role of PHB2 in parkin-mediated mitophagy was described, for the first time, in urothelial cells of cattle, naturally infected with bovine papillomavirus (BPV). The BPV2 and BPV13 E5 oncoprotein, responsible for abortive infections in urothelial cells, was detected by RT-PCR. Severe ultrastructural abnormalities of the inner mitochondrial membrane were detected using transmission electron microscopy. PHB2 formed a functional complex with PHB1. PHB2 was significantly overexpressed in mitochondrial fractions from urothelial mucosa samples taken from cattle harbouring BPV infection. PHB2 overexpression could be attributed to mitochondrial dysfunction, as its expression levels in the cytosolic, microsomal, and nuclear fractions were seen to be unmodified. Immunoprecipitation studies revealed the interaction between PHB2 and phosphorylated forms of both PINK1 and parkin. Furthermore, PHB2 interacted with LC3-II, a marker of autophagosomal membranes and autophagy receptors, such as p62 and optineurin. PHB2 was shown to interact with transcription factor EB (TFEB), which is activated following parkin-mediated mitophagy, and embryonic stem cell-expressed Ras (ERAS), a constitutive protein coded by ERas. Western blot analysis revealed a significant overexpression of unphosphorylated TFEB in mitochondrial and nuclear fractions from urothelial mucosa samples from cattle suffering from BPV infection. Finally, PHB2 interacted with ERAS, believed to be involved in mitophagosome maturation. Taken together, the molecular and ultrastructural findings of this study suggested that BPV infection is responsible for parkin-dependent mitophagy, in the pathway of which PHB2 plays a crucial role.

Bovine Papillomavirus Type 2 Infection Associated with Papillomatosis of the Amniotic Membrane in Water Buffaloes (Bubalus bubalis)

Multiple papillomatous nodules were observed scattered over the amniotic membrane in six water buffaloes that had recently aborted. Grossly, some of the nodules had multiple villous projections while others appeared as single prominent conical or cylindrical horns. Histology revealed folded hyperplastic and hyperkeratotic epithelium supported by a narrow fibro-vascular stalk. Using PCR, sequences of the bovine Deltapapillomavirus type 2 (BPV-2) E5 gene were amplified from the amniotic papillomas. Furthermore, expression of the E5 gene was detected using reverse transcription (RT)-PCR. Western blotting revealed BPV-2 E5 oncoprotein as well as L1 protein, suggesting both abortive and productive infection. Additionally, a functional complex composed of BPV-2 E5 oncoprotein and the phosphorylated PDGFβR was detected, which is consistent with the activation of PDGFβR by the interaction with BPV-2 E5 oncoprotein. These results demonstrate that BPV-2 can infect the amnion of water buffaloes and suggest that this infection may cause proliferation of the epithelial cells of the amnion. While the precise pathogenesis in uncertain, it is possible that BPV-2 infection of stratified squamous epithelial cells within squamous metaplasia foci and/or amniotic plaques could lead to papilloma formation. Papillomavirus-associated amniotic papillomas have not previously been reported in any species, including humans.

Human Papillomavirus 16 E5 Inhibits Interferon Signaling and Supports Episomal Viral Maintenance

ABSTRACT Human papillomaviruses (HPVs) infect keratinocytes of stratified epithelia. Long-term persistence of infection is a critical risk factor for the development of HPV-induced malignancies. Through the actions of its oncogenes, HPV evades host immune responses to facilitate its productive life cycle. In this work, we discovered a previously unknown function of the HPV16 E5 oncoprotein in the suppression of interferon (IFN) responses. This suppression is focused on keratinocyte-specific IFN-κ and is mediated through E5-induced changes in growth factor signaling pathways, as identified through phosphoproteomics analysis. The loss of E5 in keratinocytes maintaining the complete HPV16 genome results in the derepression of IFNK transcription and subsequent JAK/STAT-dependent upregulation of several IFN-stimulated genes (ISGs) at both the mRNA and protein levels. We also established a link between the loss of E5 and the subsequent loss of genome maintenance and stability, resulting in increased genome integration. IMPORTANCE Persistent human papillomavirus infections can cause a variety of significant cancers. The ability of HPV to persist depends on evasion of the host immune system. In this study, we show that the HPV16 E5 protein can suppress an important aspect of the host immune response. In addition, we find that the E5 protein is important for helping the virus avoid integration into the host genome, which is a frequent step along the pathway to cancer development.