3. Results
3.1 Virological findings
It is well known that bovine δPVs are the most important infectious
agents involved in the etiopathogenesis of the majority of bovine
urothelial tumours (Campo et al., 1992; Roperto et al., 2010a). E5
oncoprotein expression is correlated with the transformation of both
mesenchymal and epithelial cells to form benign and malignant tumours
(Suprynowicz et al., 2006). Therefore, we attempted to verify whether
the δPV E5 oncoprotein was expressed in the examined samples. First, we
detected E5 oncoprotein transcripts by RT-PCR, the sequencing of which
showed 100% identity with BPV-2 and BPV-13 sequences deposited in
GenBank (Accession numbers M20219.1 and JQ798171.1, respectively)
(Supplemental Fig. S1). Furthermore, Western blot analysis revealed the
expression of E5 oncoprotein, which showed that abortive infection, a
prerequisite for immune evasion and cell proliferation, takes place
(Fig. 1).
3.2 Expression of tripartite motif-containing 25 (TRIM25) and Riplet
proteins.
As many viruses, including human papillomavirus, have E3 ubiquitin
ligases as their targets (Ban et al., 2018), we wondered whether the
bovine δPV E5 oncoprotein might interact with some ligases involved in
the antiviral innate immune response mediated by RLRs, the
ubiquitination of which appears to be a key post-translational
modification. However, the molecular mechanisms of ubiquitin-mediated
RIG-I and MDA5 activation remain to be fully understood (Shi et al.,
2017; Oshiumi, 2020).
Many studies have reported that TRIM25 and Riplet are two essential E3
ubiquitin ligases for RIG-I signalling as they are known to ubiquitinate
and activate RLRs (Oshiumi et al., 2013; Oshiumi, 2020).
Therefore, we investigated these two ligases by performing
co-immunoprecipitation studies using anti-TRIM25 and anti-Riplet
antibodies. The assay revealed the presence of E5 oncoprotein in
anti-TRIM25 immunoprecipitates only, suggesting that the E5 oncoprotein
of bovine δPVs interacts with TRIM25 but not with Riplet (Fig. 2). Our
results are in line with in vitro studies performed on cells
experimentally infected with HPV18, which showed that TRIM25 but not
Riplet was a target of viral E6 oncoprotein (Chiang et al., 2018). We
then investigated the expression levels of these two ligases. Western
blot analysis of total extracts detected unmodified levels of Riplet
expression (Fig. 3) and a statistically significant reduction in the
expression of TRIM25 (Fig. 4). To understand whether the marked
reduction in TRIM25 expression levels could be attributed to
transcriptional events and/or increased protein degradation, we
investigated the presence of TRIM25 transcripts by RT-PCR. Sequencing of
the obtained cDNA amplicons showed 100% identity with bovine TRIM25
sequences deposited in GenBank (Accession number: NM_001100336.1)
(Supplemental Fig. S2). Then, we performed a real-time PCR analysis on
cDNA using specific primers for bovine TRIM25. This molecular assay did
not show any variation in transcript expression in cells infected with
bovine δPVs compared with cells from clinically normal cattle (Fig. 5).
These results suggest that bovine δPVs interfere at the protein level
rather than at the transcriptional level in reducing TRIM25 expression.
3.3. Expression levels of RIG-I and MDA5 and their downstream effectors
Expression of RLRs is ubiquitous and is typically maintained at low
levels in resting cells, but is greatly increased after virus infection
(Loo and Gale, 2011). Therefore, we decided to investigate RLR
expression during spontaneous BPV infection.
We detected reduced expression levels of both RIG-I and MDA5 by Western
blot analysis in urothelial cells infected by bovine δPVs compared with
urothelial cells from clinically normal cattle (Fig. 6). We assumed that
the levels of these proteins could be due to transcriptional reduction.
Using specific primers for bovine RIG-I and MDA5, we carried out a
real-time PCR. Sequencing of the transcript amplicons revealed cDNA
fragments showing 100% identity with bovine RIG-I and MDA5 sequences
deposited in GenBank (Accession numbers:
XM_002689480.6
and
XM_010802053.2,
respectively) (Supplemental Fig. S3). Real-time PCR of cDNA revealed a
statistically significant reduction in both RIG-I and MDA5 transcripts
in δPV-positive cells compared with δPV-negative cells (Fig. 7). These
results suggest that, like HPVs, bovine δPVs may interfere at the
transcriptional level rather than at the protein level in reducing RIG-I
and MDA5 expression to prevent their antiviral activities.
RIG-I and MDA5 interact with a mitochondrial adaptor, the mitochondrial
antiviral signalling (MAVS) protein (Yoneyama et al., 2015; Oshiumi,
2020). It remains unclear how MAVS acts as a scaffold to assemble the
signalosome in RLR-mediated antiviral signalling (Chen et al., 2018).
Western blot analysis of MAVS expression revealed unmodified protein
expression levels in both δPV-infected and healthy cells (data not
shown). Our results are in line with experimental data showing that the
expression levels of MAVS did not significantly vary in cells in which
the E6 oncoprotein of HPV18 was shown to act as a RIG-I transcriptional
repressor (Albertini et al., 2018). We then performed
co-immunoprecipitation studies using an anti-MAVS antibody. Western blot
analysis performed on the immunoprecipitates detected the presence of
RIG-I and MDA5 as well as TRIM25, phosphorylated TANK-binding protease 1
(pTBK1), phosphorylated interferon regulatory factor 3 (IRF3), and
Sec13, which is believed to be a positive regulator of MAVS (Chen et
al., 2018) (Fig. 8). Western blot analysis performed on total extracts
revealed a statistically significant reduction in the expression levels
of Sec13 in δPV-infected cells compared with cells from clinically
normal cattle (Fig. 9), which suggests that MAVS activation might be
compromised in cells spontaneously infected with bovine δPVs. MAVS
subsequently phosphorylates and activates TBK1 and IRF3, via an unknown
mechanism, which results in the production of interferons as well as
proinflammatory factors (Fang et al., 2017). Western blot analysis
performed on anti-MAVS immunoprecipitates revealed the presence of pTBK1
and pIRF3, which suggests that MAVS forms a complex with pTBK1 and pIRF3
and plays a critical role in driving and coordinating synergistic
functional activities of these downstream components. Moreover, we
investigated the expression levels of TBK1 and IRF3 in total extracts by
immunoblotting, which revealed statistically significant reduced levels
of both proteins in cells infected with bovine δPVs compared with
healthy cells (Fig. 10). Furthermore, western blot analysis revealed
statistically significant reduced expression levels of pTBK1 (Fig. 11).
TBK1 is activated via phosphorylation (Liu et al., 2015), which in turn
phosphorylates and activates IRF3. Subsequently, IRF3 enters the nucleus
to activate type 1 IFN (Fitzgerald et al., 2003; Fang et al., 2017).
Altogether, our results suggest that the transcriptional downregulation
of RIG-I and MDA5 in cells infected with bovine δPVs is responsible for
an aberrant downstream signalling pathway, including TBK1/IRF3, which
may lead to the impairment of the host antiviral response. Because of
downregulated RLRs, an adequate innate immune response is not elicited
against spontaneous bovine δPV infection, thus leading to persistent
infection in the cells.
4. DISCUSSION
This study provides novel mechanistic insights into the role of E5
oncoprotein in dysregulating the host antiviral innate immune response
in a spontaneous model of bovine papillomavirus disease. Our study
showed, for the first time, that the E5 oncoprotein of bovine δPVs
interacts with TRIM25, a key player in antiviral immunity (Koliopoulos
et al., 2018), to hamper innate immune signalling pathway mediated by
RIG-I and MDA5. These results are of interest as there are very limited,
controversial in vivo studies based on the role of TRIM25 in RLR
activation, which remains elusive (Hayman et al., 2019; Wang and Hur,
2020).
E5 oncoprotein did not appear to influence the transcriptional activity
of TRIM25; therefore, it is conceivable that E5 oncoprotein enhanced
TRIM25 proteasomal degradation, which may hinder the activation of RIG-I
and MDA5. It is well known that TRIM25 ubiquitinates and activates RLRs
in a dose-dependent manner (Gack et al., 2007). Our results appear to be
corroborated by experimental studies that showed that HPV oncoproteins
could enhance the proteasomal degradation of TRIM25 (Chiang et al.,
2018). Furthermore, our study suggested the existence of multiple
evasion mechanisms based on bovine δPV-mediated inhibition of key
components of the RLR pathways. Indeed, E5-expressing cells showed a
marked reduction in the transcriptional activity of both RIG-I and MDA5.
Reduced RIG-I and MDA5 mRNA levels detected by real-time PCR suggested
that some proteins of bovine δPVs could downregulate the transcriptional
activity of RIG-I and MDA5, which allowed δPVs to impair the innate
antiviral response, a prerequisite for persistent infection. Our results
appeared to be strengthened by experimental data from in vitrostudies in which HPV oncoproteins have been shown to act as
transcriptional repressors of RIG-I and MDA5 to impair the viral host
response during persistent infection (Reiser et al., 2011; Albertini et
al., 2018). RLRs catalyse the conversion of MAVS fibrils to prion-like
aggregates. Although MAVS activation is a complex, multistep process,
this conformational change of MAVS is essential for the recruitment of
downstream signalling molecules (Hou et al., 2011). Not much is known
about the mechanism(s) of how MAVS functions in antiviral signalling
pathways (Chen et al., 2018); therefore, the activation mechanism of
MAVS downstream pathways remains elusive (Zhu et al., 2019). In our
study, MAVS expression levels did not vary significantly. Many viruses
block RLR-mediated immune signalling thus inhibiting host antiviral
response without modifying MAVS expression levels (Zhang et al., 2020).
It is conceivable that in our spontaneous model of PV infection, the
marked reduction in the expression levels of RIG-I and MDA5 may be
responsible for the loss of conformational changes thus compromising the
activation of MAVS, which is necessary for activating and propagating
the antiviral signalling cascade. In addition, we found reduced
expression levels of Sec13, which may contribute to further attenuation
of MAVS downstream signalling. It has been suggested that Sec13
facilitates MAVS aggregation and ubiquitination and is thus required for
RLR-MAVS-related antiviral responses (Chen et al., 2018). It has been
shown that Sec13 expression correlates with MAVS activation. Indeed, the
overexpression of Sec13 increases MAVS activation, whereas Sec13
downregulation attenuates MAVS activation (Chen et al., 2018). In
vitro studies have shown that MAVS may serve as a scaffold to
facilitate the interaction of TBK1 with IRF3 (Liu et al., 2015). MAVS
has been shown to activate the transcription factor IRF3 through TBK1
(Fang et al., 2017). We found a marked reduction in the expression
levels of total and phosphorylated TBK1, which may result in
perturbation of IRF3 activation as TBK1 plays a crucial role in allowing
efficient IRF3 phosphorylation in the IFN-producing pathways that
require MAVS as the adaptor protein (Fang et al., 2017). Many viruses
inhibit RIG-I/MAVS signalling by blocking TBK1 phosphorylation (Darlympe
et al., 2015). It is conceivable that the E5 oncoprotein of bovine δPVs
is a key player involved in the downregulation of TBK1 activation. Low
expression of TBK1 has been shown to markedly reduce IFN1 induction
(Seth et al., 2005) and proinflammatory macrophage (M1) polarisation
(Stone et al., 2019). Furthermore, we found reduced expression levels of
IRF3, which may hamper their interaction network, a critical step in the
production of IFNs (Ding et al., 2014; Liu et al., 2015).
Bovine δPVs must escape innate immune surveillance to establish
persistent infections and viral proteins may manipulate this process
through several mechanisms. This study showed that similar to human PVs,
bovine PVs perturb the RLR-mediated innate immune signalling pathway
through the viral E oncoprotein, which is encoded in the early stages of
PV infection. This perturbation results in an abnormal host antiviral
response, which allows PVs to continue their infectious cycle leading to
persistent viral infection. Bovine δPVs reduce the levels of the DNA
sensors that can recognise BPVs, which can hamper pTBK1 signalling as
well as the production of IFNs, similar to human PVs (Hong and Laimins,
2017). IFN production plays a crucial role in the immune response
against PV infection as IFNs promote the clearance of latent PV episomes
in persistently infected cells (Westrich et al., 2017) and/or rapid
reduction in PV episome copies per cell (Herdman et al., 2006). It has
been suggested that basal cells in the initial infection usually contain
low levels (around 100 copies per cell) of human and bovine PV episomes
(Turek et al., 2002; Groves and Colemans, 2015). Animal cells that fail
to resolve their infection and retain oncogene expression for years can
facilitate tumourigenesis by BPVs (Doorbar, 2006).
In conclusion, bovine δPVs must escape innate immune surveillance to
establish persistent infections, and viral proteins manipulate this
process through several mechanisms. Despite the importance, molecular
mechanisms for many bovine δPV oncoproteins remain poorly characterised,
in part due to challenges in identifying their substrates. Therefore,
further investigations aimed to clarify the functional role of viral
oncoproteins at the intersection of immune evasion and aberrant
proliferation of cells persistently infected by bovine δPVs, warrant
future research.