RIOK3-Mediated Akt phosphorylation facilitates synergistic replication of Marek’s disease and reticuloendotheliosis viruses

ABSTRACT Co-infection of Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) synergistically drives disease progression, yet little is known about the mechanism of the synergism. Here, we found that co-infection of REV and MDV increased their replication via the RIOK3-Akt pathway. Initially, we noticed that the viral titres of MDV and REV significantly increased in REV and MDV co-infected cells compared with single-infected cells. Furthermore, tandem mass tag peptide labelling coupled with LC/MS analysis showed that Akt was upregulated in REV and MDV co-infected cells. Overexpression of Akt promoted synergistic replication of MDV and REV. Conversely, inhibition of Akt suppressed synergistic replication of MDV and REV. However, PI3K inhibition did not affect synergistic replication of MDV and REV, suggesting that the PI3K/Akt pathway is not involved in the synergism of MDV and REV. In addition, we revealed that RIOK3 was recruited to regulate Akt in REV and MDV co-infected cells. Moreover, wild-type RIOK3, but not kinase-dead RIOK3, mediated Akt phosphorylation and promoted synergistic replication of MDV and REV. Our results illustrate that MDV and REV activated a novel RIOK3-Akt signalling pathway to facilitate their synergistic replication.


Introduction
Both Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) are important oncogenic viruses that cause immunosuppression and tumours in chicken flocks, leading to significant economic losses in the poultry industry [1,2]. In addition to singleinfection, numerous studies have reported the simultaneous infection of MDV and REV in chicken flocks [3][4][5][6][7][8][9][10][11][12][13][14][15]. Co-infection of MDV and REV alters the biological characteristics, pathogenicity, and epidemiologic status of the two viruses and modulates the immune response and host susceptibility. The integration of the partial or full REV genome into MDV is a common phenomenon in MDV and REV co-infected cells [16][17][18]. These recombination events can alter the biological functions of MDV and REV [19][20][21][22][23][24][25], which could promote the transmission and pathogenicity of the two viruses [26][27][28][29]. Furthermore, MDV and REV co-infection significantly enhance disease severity and decrease the antibody levels elicited by MD vaccines, consequently increasing susceptibility to secondary infections [30,31]. In addition, REV can be transmitted by inoculation with contaminated MD vaccines [32][33][34][35]. Despite MDV and REV co-infection events in the poultry being increasingly detected, little is known about the synergistic mechanism of the two viruses.
It has been demonstrated that Akt activity modulated by many viruses for replication function. HIV inhibit premature apoptosis by inducing Akt activity to facilitate virus replication, and herpes simplex virus 1 (HSV-1) replication also benefit from Akt phosphorylation [42,43]. In viral co-infection, HIV Nef synergizes with Kaposi's sarcoma-associated herpesvirus (KSHV) vIL-6, which results in the activation of the Akt pathway, enhancing angiogenesis and tumorigenesis [38]. For some viruses, the activation of Akt, but not PI3k, plays an important role in viral replication. Akt phosphorylates the phosphoprotein of nonsegmented negative-strand RNA viruses, driving RNAdependent RNA polymerase activity [44,45].
RIOK3 (right open reading frame kinases 3) is a conserved atypical serine/threonine protein kinase within the RIO kinase family. It was reported that RIOK3 is an oncogene in breast cancer, glioma, pancreatic cancer and prostatic cancer through a variety of regulatory mechanisms [46,47]. RIOK3 was also found to regulate the type I IFN pathway during viral infection [48] and play as a component of pre-40S preribosomal particles [49].
A recent study demonstrated that MDV activates the PI3K/Akt pathway, leading to reduced host cell apoptosis and increased virus replication [50]. However, there is no information on the role of Akt activation in MDV and REV co-infected host cells. Here, we reported that the Akt is more obviously activated and sustained in MDV and REV co-infected cells compare with in single virus infected cells. Furthermore, we revealed that RIOK3, but not PI3K, boosted Akt activity to promote the synergistic replication of MDV and REV, and we determined that the kinase activity of RIOK3 is required for the interaction between RIOK3 and Akt.
Pretreatment with the PI3K inhibitor LY294002 and Akt inhibitor MK2206 (Beyotime Biotechnology) was conducted at optimum concentrations to avoid affecting CEF viability.

Experiment design of REV and MDV co-infection
We used three formats to establish the optimal coinfection model: initial infection with MDV then with REV 24 hours later; simultaneous MDV and REV infection; initial infection with REV then with MDV 24 hours later. The cell status was monitored, and the virus growth curve was evaluated using plaqueforming unit (PFU) and the 50% tissue culture infective dose (TCID 50 ). The first two models caused cell death within 48 hours post-infection (hpi), and the two viruses did not show significant synergism. The third infection model showed significant synergistic replication of MDV and REV, and cell death occurred 72 hpi. Therefore, we selected the third co-infection model for subsequent experiments (Figure 1(a)). The optimal multiplicity of infection (MOI) was chosen to allow virus replication while causing minimal damage to CEF cells. Confocal imaging and western blotting were used to determine viral infection and proliferation. The mock, MDV-and REV-infected, and MDV +REV co-infected CEF cells were prepared for comparative TMT-LC-MS/MC analysis at the appropriate time intervals. Each sample comprised three technical replicates, and each experiment was conducted thrice.

MDV and REV replication analyses
The replication of MDV and REV was measured using the PFU and TCID 50 methods in the CEF cells at various time points, respectively. Briefly, 100 PFU of Md5 strain were inoculated into the CEF cells in 6-well plates and incubated at 37 °C with 5% CO 2. The virusinfected CEFs were collected from 24 to 108 hpi to determine MDV replication in CEF cells, a series of two-fold dilutions was prepared into 96-well plates containing the CEFs in triplicate. Thereafter, count the number of plaques to determine viral titres from three independent experiments. 1000 TCID 50 of REV SNV were inoculated into the CEF cells in 6-well plates. Infected cell cultures were harvested at 24, 48, 72, 96,108 hpi. The TCID 50 per millilitre of REV was determined by an immunofluorescence assay, using the Reed-Muench formula. The infectious progeny were subsequently harvested in triplicate from REVinfected cell cultures.
The MDV and REV genome copy numbers was measured by real-time quantitative PCR (qPCR) as previously described [29,31].

Confocal imaging
Cultured CEF cells and DF-1 cells were single infected or co-infected with MDV and REV in 15-mm culture dishes. DF-1 cells were transfected with the RIOK3-Flag, empty vector plasmid or the Akt-HA plasmid. For confocal imaging, firstly, cells were washed with cold PBS, and then cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.2% Triton X-100 for 15 minutes. Next, cells were blocked with 5% BSA for 1 h. Thereafter, the CEF cells were incubated with mouse anti-REV-gp90, FITC-labelled goat anti-mouse IgG or rabbit anti-MDV-pp38, Cy3labelled goat anti-rabbit IgG diluted in PBS for 1 h. For DF-1 cells, mouse anti-Flag or rabbit anti-HA antibodies and FITC-labelled goat anti-rabbit IgG Cy3labelled goat anti-mouse IgG secondary antibodies (BIOSS) were used. The overlapping of the two fluorescent marker colours appeared yellow. In addition, the nuclei of all infected cells were stained using DAPI (Beyotime Biotechnology). After washing five times with PBS, we examined the cells subsequently using an SP8 confocal laser scanning microscope (CLSM; Leica Microsystems, Wetzlar, Germany).

Immunoprecipitation (IP) and western blot (WB) assay
IP was performed with cell lysates isolated from DF-1 cells. Briefly, DF-1 cells were cultured in 6-well plates one day and then transfected with the indicated plasmids. The cells were removed from the medium at 48 h post-transfection, washed with cold PBS, and then directly lysed on the plate with Lysis/Equilibration Buffer (TaKaRa). After centrifugation at 1000× g for 5 minutes, the supernatants were incubated overnight with the indicated antibodies at 4 °C. Thereafter, 20 μL protein G-sepharose beads (Roche Holding AG, Basel, Switzerland) were added to the sample. After incubated, the beads were washed five times with PBS, transferred to Eppendorf tubes with SDS loading buffer, and then boiled for 10 minutes before western blotting analysis.
For western blotting, cells were washed three times with PBS, lysed on ice with NP-40 lysis buffer (Beyotime Biotechnology). The samples were subsequently separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Afterwards, the membranes were blocked using QuickBlockTM Blocking Buffer (Beyotime Biotechnology) for 15 minutes and incubated overnight with the indicated primary antibodies at 4 °C. The membranes were washed five times with Tris-buffered saline with Tween 20 and incubated secondary antibodies.

HPLC fractionation and LC-MS/MS analysis
Cells infected with MDV and/or REV and the Akt-Flag IP samples were analysed using a high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific, Waltham, MA, USA) with an Agilent Zorbax 300Extend-C18 column. The tryptic peptides were dissolved on an EASY-nLC 1000 UPLC system (450 nL/min). Tandem mass spectrometry (MS/MS) was performed using a Q Exactive TM HF-X system (Thermo Fisher Scientific). The MS/MS data were searched in the Uniprot-gallus FASTA database using the Maxquant search engine.
For protein abundance ratios, a 1.2-fold change was taken as the threshold, and a corrected p-value <0.05 was adopted to identify significant changes. To annotate protein pathways, Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (http://www.genome. jp/kegg/tool/map_pathway2.html) was used.

In vivo experiment
1-day-old SPF White Leghorn chickens were purchased from the poultry institute, Shandong academy of agricultural science. The 120 birds were randomly numbered and divided into four groups, then individually housed in negative pressure-filtered air isolators. On day one, The first group was inoculated with 2000 PFU of MDV in 200 µL diluent, while the second group was inoculated with 10 4 TCID 50 REV in 200 µL diluent. The third group was treated as follows: on day one, 2000 PFU of MDV in 200 µL diluent; on day four, 10 4 TCID 50 REV in 200 µL diluent. 30 chickens in control group were injected with DMEM. On 3, 7, 14, 21 and 35 days post-infection (dpi), four birds were randomly selected from each group and humanely euthanized. After necropsy, the spleen were collected for viral copies and Akt/p-Akt expression analysis. The DNA and RNA was extracted using the TIANGEN kit (TIANGEN, Beijing, China) and detected by qPCR. Animal experiments were conducted following protocols approved by the Committee on the Ethics of Animal Experiments of the Shandong Agricultural University.

Statistical analysis
Statistical significance among groups was determined by one-way repeated measures ANOVA, and the data were presented as the means ± SD. Statistical significance was set at p-value <0.05.

MDV and REV facilitate mutual replication and activate the Akt pathway in vitro
To determine the effects of MDV and REV co-infection on their replication in vitro, CEF cells were infected with REV (MOI = 1, 1000 TCID 50 ) and then with MDV (MOI = 0.1,100 PFU) after 24 hours as described in the experimental design (Figure 1(a)). The viral titres of REV and MDV were quantified using TCID 50 and the plaque assays from 24 to 108 hpi. FITC-labelled anti-gp90 and Cy3-labelled anti-pp38 antibodies were used to detect REV-gp90 and MDV-pp38 expression and localization by confocal laser scanning microscopy. The results indicated that the replication rate of REV or MDV was higher at 24 hpi (p < 0.05), 48 hpi (p < 0.01) and 72 hpi (p < 0.05) in the REV and MDV coinfected group compared with that in the MDV infected control group (24 hpi, 48 hpi, 72 hpi) or REV infected control group (48 hpi, 72 hpi, 96 hpi). The replication rate of MDV or REV was lower at 96 hpi and 108 hpi in REV and MDVco-infected cells (Figure 1(b)). The genome copy numbers of REV and MDV were measured by qPCR from 24 to 108 hpi. The results showed that the replication rate of REV or MDV was higher from 24 to 96 hpi and lower at 108 hpi in REV and MDV co-infected cells ( Figure S1). Furthermore, viral protein expressions were evaluated by western blotting at 48 hpi, and the results showed that the protein expression levels of the gp90 of REV and pp38 of MDV in co-infected cells were significantly higher than those in single infected cells (Figure 1(c)). Meanwhile, the highly expressed gp90 and pp38 were co-localized in the cytoplasm of REV and MDV coinfected cells 48 hpi (Figure 1(d)). To determine which signalling pathway is regulated in the process of REV and MDV synergistic replication, the REV and MDV co-infected CEF cells 48 hpi, MDV-infected CEF cells 48 hpi and REV-infected CEF cells 72 hpi were selected for further TMT-LC-MS/MS analysis. KEGG and heatmap analysis showed that the Akt pathway was enriched (Figure 1(e)) and Akt was significantly upregulated (Figure 1(f), Supplementary Table S1, p ≤ 0.05, ratios ≥1.2 or ≤0.83) in REV and MDV co-infected cells. All the results suggested that REV and MDV synergistically increased viral replication and upregulated Akt pathway in vitro.

Akt promotes synergistic replication of MDV and REV
To determine the effect of Akt on the synergistic replication of MDV and REV, Akt overexpression and interference were performed in CEF. CEF cells were transfected with Akt-Flag followed by infection with REV and/or MDV 24 h later, and the viral titres were determined by plaque and TCID 50 assays 48 hpi. Akt overexpression remarkably enhanced the synergistic replication of MDV and REV (Figure 2(a,b,d)), while Akt knockdown significantly suppressed their synergism (Figure 2(a,c,d)).

High Akt phosphorylation level is responsible for MDV and REV synergistic replication, independent of PI3K
To determine how the Akt pathway affected the synergistic replication of MDV and REV, the expression and phosphorylation level of Akt and 4EBP1 were evaluated by western blotting. Cell lysates of the mock-, singleand co-infected CEF cells 6 and 48 hpi were prepared for western blotting. To determine viral infection, the pp38 (MDV) and gp90 (REV) protein expression levels were also evaluated by western blotting. The results revealed that phosphorylation status of Akt was increased in MDV/REV infected cells compared to the Akt phosphorylation levels in noninfected cells. Simultaneously, the phosphorylation level of Akt was increased in REV and MDV co-infected cells relative to that in single-infected cells 6 hpi (Figure 3(a,c)) and 48 hpi (Figure 3(b,c)). Concomitantly, the downstream Akt target 4EBP1 was more strongly phosphorylated in co-infected cells. The high levels of Akt phosphorylation subsequently persisted throughout both time points in REV and MDV co-infected cells while remaining undetectable in single-infected cells ( Figure 3(a,b)). These data indicated that Akt activation was transient in MDV or REV single-infected cells but was sustained in REV and MDV co-infected cells. All the results suggested that MDV and REV synergistically upregulated Akt pathway in vitro.
To investigate whether Akt activation by MDV and REV was critical for the replication of the two viruses, CEF cells pretreated with or without Akt inhibitor MK2206 were infected with MDV and/or REV, and then the virus titres were determined 48 hpi. The CCK-8 assay showed that the optimum MK2206 concentration not affecting CEF viability was 25 nM (Figure 4(e)). Cell lysates were prepared to determine Akt and 4EBP1 phosphorylation 6 hpi using western blotting. Both Akt and 4EBP1 phosphorylation were decreased in MDV/REV infected and co-infected cells, indicating that the activation of Akt plays a critical role in the replication of the two viruses (Figure 4(a,  g)). Furthermore, the replication of MDV or REV was significantly inhibited in both the single-and coinfected cells (Figure 4(c,d)). These results indicated that high Akt phosphorylation level promoted the synergistic replication of REV and MDV in coinfected cells.
To further understand the effect of PI3K, the major upstream molecule of Akt, on REV and MDV synergistic replication, we evaluated Akt and 4EBP1 phosphorylation after PI3K inhibition. The mock-, single-, and co-infected cells were maintained in a standard medium differentially treated with a specific PI3K inhibitor, LY294002. The CCK-8 assay showed that the optimum LY294002 concentration not affecting CEF viability was 20 µM (Figure 4(f)). Cell lysates were prepared 6 hpi and analysed by western blotting. On the one hand, in single-infected cells, Akt and 4EBP1 phosphorylation and the downstream readout of Akt activity were not detected upon LY294002 treatment (Figure 4(b,g)), confirming the efficacy of the inhibitor. On the other hand, the level of Akt and 4EBP1 phosphorylation in co-infected cells was reduced but not eliminated relative to that of single-infected cells. Furthermore, the replication of MDV or REV was not significantly inhibited in the co-infected cells, while virus replication was significantly reduced in REV and MDV single-infected cells 48 hpi (Figure 4(c,d,g)). These results indicated that PI3K inhibition did not influence viral growth in REV and MDV co-infected cells.

RIOK3 is required for synergistic replication of MDV and REV
To further investigate the host factors responsible for Akt activation in REV and MDV co-infected cells, we performed IP-MS/MS to identify potential Aktassociated proteins. A construct containing the Akt gene and an empty vector were transfected into CEF cells which were then infected with MDV or/and REV. Thereafter, cells were subjected to lysis for the IP procedure using anti-Flag affinity gel 24 hpi, followed by 10% SDS-PAGE and MS analysis. The proteins present only in the IP product from REV and MDV co-infected cells but not in the IP product from MDV or REV infected cells were selected -we screened several proteins in the IP product treated with Akt. Of the screened proteins, a serine/threonine-protein kinase, RIO kinase 3 (RIOK3), attracted our attention because it was not only identified as an Aktinteracting protein in REV and MDV co-infected   (a and b). The data represent the mean ± SD of three independent experiments. One-way ANOVA, (*, p <0.05; **, p <0.01).
cells, but it was also significantly upregulated in REV and MDV co-infected cells (Figures 5(a,e) and 1(f)). To investigate whether RIOK3 was critical for the Akt phosphorylation and the replication of the two viruses in REV and MDV co-infected cells, CEF cells were transfected with eukaryotic expression plasmids expressing shRIOK3 and then infected with REV and MDV. The level of Akt phosphorylation was subsequently determined by western blotting 48 hpi. Virus titres were determined by the plaque and TCID 50 assays 24, 48, 72, 96 and 108 hpi. The results showed that RIOK3 knockdown significantly decreased the level of Akt phosphorylation ( Figure 5(b,f)) and suppressed the synergistic replication of REV and MDV in co-infected cells ( Figure 5(c,d)). These data indicated that REV and MDV synergistically upregulated RIOK3 expression in vitro.

RIOK3 increases Akt phosphorylation
Since RIOK3 is a kinase, it is possible to investigate the relevance of the association between the RIOK3 and Akt activity. To examine the role of RIOK3-mediated knockdown attenuates synergistic replication of REV and MDV. CEF cells were transfected with shRIOK3 and then co-infected with REV and MDV for 48 h. The MDV titre was measured using plaque quantification (c) and REV titre detected by TCID 50 (d). (e and f) Quantification of relative RIOK3 band intensities to actin and relative pAkt band intensities to Akt (a and b). The data represent the mean ± SD of three independent experiments. One-way ANOVA, (*, p <0.05; **, p <0.01).
Akt phosphorylation in gene regulation, we transfected the constructed RIOK3 recombinant vector to assess Akt activity. Furthermore, to determine whether the kinase activity of RIOK3 is required for Akt phosphorylation, we constructed a RIOK3 kinase-dead mutant, K290A, in which the invariant lysine in subdomain II that is critical for ATP binding was mutated. The results showed that RIOK3 activated Akt activity in a dose-dependent manner (Figure 6(a,b,e)). As shown in Figures 6(c,d), RIOK3 overexpression enhanced the replication of REV and MDV. However, RIOK3-K290A did not affect virus replication in MDV-or REVinfected cells, suggesting that the kinase activity of RIOK3 is important for Akt phosphorylation and viral replication.

RIOK3 interacts with Akt
To further investigate whether RIOK3 mediates the Aktpromoted synergistic replication of REV and MDV, we examined the physical association between RIOK3 and Akt. Cellular lysates from DF-1 cells co-transfected with RIOK3-Flag, RIOK3-K290A-Flag and Akt-HA were subjected to immunoprecipitation. Interestingly, our results demonstrated that Akt was efficiently co-precipitated with RIOK3-Flag, while failed associated with kinase- dead RIOK3-K290A, and reciprocally, RIOK3 could also be immunoprecipitated by Akt. These results suggested that the kinase activity of RIOK3 is important for its interaction with Akt (Figure 7(a,b)). In addition, to verify the interaction of RIOK3 with Akt, we examined the localization of RIOK3 and Akt in DF-1 cells by confocal microscopy, and the results revealed that Akt co-localized with RIOK3 in the cytoplasm (Figure 7(c)).

RIOK-Akt pathway is activated in MDV and REV co-infected chicken
To determine whether MDV and REV facilitate mutual replication and activate the RIOK3-Akt pathway in vivo, we first determined the replication curves of MDV and REV in MDV/REV infected or co-infected chicken spleen. As shown in Figure 8(a,b), the replication rate of MDV or REV was higher from 3 dpi to 21 dpi in the REV and MDV co-infected chicken spleen compared with that in the MDV/REV infected control group and reached a peak at 14 dpi. Furthermore, western blotting analysis showed that the protein levels of RIOK3, Akt, p-Akt and p-4EBP1 in the spleen of REV and MDV co-infected chicken spleen were significantly higher than in those of REV/MDV infected chicken spleen at 14 dpi, respectively (Figure 8(c,d)).
These data indicated that MDV and REV synergistically increased viral replication and activated RIOK3-Akt pathway in vivo.

Discussion
Viruses usually activate intracellular PI3K/Akt signalling to promote viral infection and replication [36,37,39,40]. The activation of Akt can provide the benefits of increasing growth and suppressing apoptosiss [40]. Generally, the expression of Akt is strictly regulated in cells [52,53]. However, for many DNA or RNA viruses, viral infection benefit from increased Akt or phosphorylated Akt expression level. This phenomenon also occurs in more than one virus co-infected host. For example, It has reported that HIV Tat activates PI3K/Akt signalling and potentiates KSHV proteins oncogenic activity in KSHV and HIV co-infected hosts [54][55][56]. However, whether the synergistic replication between REV and MDV is regulated by PI3K/Akt pathway remains unclear.
In the present study, we observed that REV and MDV co-infection enhanced mutual replication in vitro and in vivo, indicating synergism between MDV and REV. To investigate whether the PI3K/Akt pathway was involved in this synergism, we conducted TMT-LC/MC analysis [51]. Heatmap and KEGG analysis showed that Akt protein levels were significantly upregulated, and Akt was involved in the main signalling pathway in REV and MDV co-infected cells. It has been proposed that the Meq protein of MDV could interact with PI3K, activating the PI3K/Akt pathway [50]. However, no reports have demonstrated the relationship between REV replication and the PI3K/Akt signalling pathway. Here, we demonstrated that high Akt expression was considerably associated with synergistic viral replication, and silencing Akt inhibited the synergistic replication of MDV and REV. Furthermore, we revealed that REV and MDV coinfection led to very strong and persistent Akt phosphorylation compared with the transient Akt phosphorylation observed in single-infected cells. We also revealed higher level of Akt, phosphorylation of Akt and 4EBP1 in REV and MDV coinfected chicken, indicating that Akt pathway activation likely supports synergistic viral replication of MDV and REV in vitro and in vivo.
It is well known that PI3K in virus-infected cells is sufficient to activate Akt [57,58]. For example, VP11/12 protein of herpes simplex virus 1 activates the PI3K/ Akt transient phosphorylation [59] and influenza A virus NSP1 directly binds to the P85 subunit of PI3K activates Akt pathway [60]. However, CMV protein directly targets mTOR but not directly activate PI3K or Akt to support viral replication [61]. In the present study, PI3K inhibition had little influence on MDV/REV replication in co-infected cells, indicating that PI3K is not required for the synergistic replication of MDV and REV via Akt activation. Therefore, the host molecule/s mediating Akt activation in REV and MDV co-infected cells remain to be identified. To this effect, MS and Co-IP analysis revealed an interaction between RIOK3 and Akt.
RIOK3, a conserved atypical kinase, belongs to the RIO family, including three RIO kinases: RIOK1, RIOK2 and RIOK3 [62]. It has been shown that RIOK1 and RIOK2 play a crucial role in cell cycle progression [47,62], and RIOK3 is important for autophosphorylation [63]. In general, the phosphorylation and activation of Akt are thought to be mediated by PI3K activity. Therefore, we hypothesized that Akt is a substrate for RIOK3. As shown in the present study, the overexpression of RIOK3, but not of the kinasedead mutant, induced Akt phosphorylation. Furthermore, induction of RIOK3-mediated Akt phosphorylation resulted in activation of 4EBP1 expression, indicating that RIOK3 alone can modulate cellular signalling pathways by activating Akt. We speculated that RIOK3 and Akt co-localize in co-infected cells since immunofluorescence and co-immunoprecipitation results demonstrated that RIOK3 and Akt were corecruited.
Taken together, our results illustrate that MDV and REV activated a novel RIOK3-Akt signalling pathway to facilitate their synergistic replication. Further studies are needed to map the RIOK3 phosphorylation site of Akt to further explore the role of RIOK3-Akt pathway in viral replication, pathogenesis, and tumorigenesis during co-infection of MDV and REV.