Porcine Reproductive and Respiratory Syndrome Virus Adapts Antiviral Innate Immunity via Manipulating MALT1

ABSTRACT To fulfill virus replication and persistent infection in hosts, viruses have to find ways to compromise innate immunity, including timely impedance on antiviral RNases and inflammatory responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen causing immune suppression. MALT1 is a central immune regulator in both innate and adaptive immunity. In this study, MALT1 was confirmed to be induced rapidly upon PRRSV infection and mediate the degradation of two anti-PRRSV RNases, MCPIP1 and N4BP1, relying on its proteolytic activity, consequently facilitating PRRSV replication. Multiple PRRSV nsps, including nsp11, nsp7β, and nsp4, contributed to MALT1 elicitation. Interestingly, the elevated expression of MALT1 began to decrease once intracellular viral expression reached a high enough level. Higher infection dose brought earlier MALT1 inflection. Further, PRRSV nsp6 mediated significant MALT1 degradation via ubiquitination-proteasome pathway. Downregulation of MALT1 suppressed NF-κB signals, leading to the decrease in proinflammatory cytokine expression. In conclusion, MALT1 expression was manipulated by PRRSV in an elaborate manner to antagonize precisely the antiviral effects of host RNases without excessive and continuous activation of inflammatory responses. These findings throw light on the machinery of PRRSV to build homeostasis in infected immune system for viral settlement.

regulating MALT1 inflammatory responses were explored. This study revealed a new mechanism of PRRSV against the innate immune defense by manipulating MALT1 to antagonize anti-PRRSV RNases and alleviate inflammatory responses.

RESULTS
N4BP1 and MCPIP1 inhibit PRRSV replication, relying on their RNase activity. Expression levels of the two RNases (N4BP1 and MCPIP1) upon PRRSV infection were detected in quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis. The results showed that PRRSV infection induced RNase expression rapidly in Marc-145 cells and porcine alveolar macrophages (PAMs), especially before 36 h postinfection ( Fig. 1A and B). However, the expression levels decreased during the late stage of infection. These results indicated that host RNases N4BP1 and MCPIP1 were rapidly induced by PRRSV infection.
To assess the impact of N4BP1 on PRRSV replication, Marc-145 cells were transfected with N4BP1 expression plasmids or empty vector control and then infected with PRRSV. Virus replication levels were detected over a 36-h infection course in qRT-PCR and Western blot analysis with infected culture supernatants. As shown in Fig. 1C, the expression of PRRSV N protein was significantly inhibited in both mRNA and protein levels after overexpressing N4BP1 compared to the vector control. In addition, the virus titers in culture supernatants also were reduced after N4BP1 overexpression. The suppressive effects of MCPIP1 on PRRSV infection were demonstrated in our recent study (12). In addition, to evaluate whether N4BP1 has a synergistic effect with MCPIP1 in inhibiting PRRSV, Marc-145 cells were transfected with MCPIP1 or N4BP1 plasmids, respectively, or cotransfected with the two plasmids. The results of qRT-PCR, virus titers, and Western blot analysis showed that cotransfection of MCPIP1 and N4BP1 has a more significant suppressive effect on PRRSV replication than either single transfection (Fig. 1D). These results demonstrated the inhibitory activity of N4BP1 and MCPIP1 against PRRSV replication and the cooperative antiviral effects of these two RNases.
To further investigate whether N4BP1 and MCPIP1 antagonize PRRSV infection via their RNase domains, mutants of pig-derived N4BP1 and MCPIP1 with deficiency of RNase activity were constructed. Asp623 of human N4BP1 has been proven as the essential site for its catalytic center, and N4BP1 D623N mutant fully abrogated its RNase activity on HIV-1 mRNA (13). According to the amino acid sequence alignment of RNase domain of pig and human N4BP1, their RNase domains are highly conserved, and D624 of porcine N4BP1 is consistent with D623 of human N4BP1. Therefore, a point mutation of D624N of porcine N4BP1 was constructed ( Fig. 1E). On this basis, the antiviral effects against PRRSV of wild-type (WT) N4BP1 and D624N mutant were compared. The results of PRRSV N mRNA and protein levels, along with virus titers, showed that D624N mutant was unable to suppress PRRSV replication in 3D41CD163 cells at 24 hpi (Fig. 1F). A similar mutation was induced to porcine MCPIP1. The RNase domain structure and primary amino acid sequence of N4BP1 are similar to those of MCPIP1 (13). As indicated in the comparison of RNase domain amino acid sequences between porcine N4BP1 and MCPIP1, D624 of porcine N4BP1 and D125 of porcine MCPIP1 are homologous sites (Fig. 1G). Hence, MCPIP1 D125N mutant was constructed and assessed for the inhibitory effects on PRRSV. The results showed that wild-type MCPIP1 significantly suppressed PRRSV replication, whereas D125N mutation abolished the anti-PRRSV activity in MCPIP1 (Fig. 1H). Taken together, these results verified N4BP1 and MCPIP1, the two host RNases, suppressed PRRSV replication via their RNase activity.
MALT1 protease degrades N4BP1 and MCPIP1, facilitating PRRSV replication. Previous studies confirmed that in T cells, MALT1 protease cleaves N4BP1 and MCPIP1 (13,22). Here, to verify the effects of MALT1 on the expression of the two RNases in Marc-145 and 3D4 cells, MALT1 overexpression and knockdown assays were performed. As shown in Fig. 2A and B, monkey-or pig-derived MALT1 was overexpressed in Marc-145 or 3D4 cells, respectively, causing a significant decrease in N4BP1 and MCPIP1 protein expression. Consistent with this, knockdown of MALT1 by siRNA in Marc-145 and 3D4 cells markedly upregulated the protein levels of the two RNases ( Fig. 2C and D). Furthermore, PRRSV replication levels were determined in MALT1 overexpressed or downregulated Marc-145 cells. The results showed that MALT1 overexpression significantly upregulated virus titers in the  (Fig. 2E). On the other hand, knockdown of MALT1 by short interfering RNA (siRNA) dramatically downregulated viral replication levels (Fig. 2F).
To further clarify whether proteolytic activity of MALT1 functions in antagonizing RNase expression, Mi-2, a specific MALT1 inhibitor, was used. Mi-2 can covalently bind to the caspase-like domain of MALT1 and suppress its protease function (18). The results showed that different concentrations of Mi-2 (0 to 2 mM) markedly upregulated N4BP1 and MCPIP1 protein expression in a dose-dependent manner in Marc-145 cells (Fig. 3A) as well as in 3D4 cells (Fig. 3B). Similarly, protein levels of the two RNases increased upon Mi-2 treatment in PAMs in a dose-dependent manner (Fig. 3C). Importantly, Mi-2 treatment significantly downregulated PRRSV replication levels in Marc-145 cells and PAMs during a 36-h infection course ( Fig. 3D and E).
C472 of mouse MALT1 in its caspase-like domain has been demonstrated as the vital site to maintain its proteolytic activity (22). In porcine MALT1, C489 is homologous to C472 of mouse-derived protein (Fig. 3F). Thus, C489A mutant of pig MALT1 was constructed and used to transfect 3D4 cells. The results of Western blot analysis showed that N4BP1 and MCPIP1 protein expression was significantly downregulated after MALT1 overexpression, whereas C489A had no regulatory effects on their expression (Fig. 3G). Moreover, quantitative PCR (qPCR) and virus titer detection revealed that MALT1 overexpression notably upregulated PRRSV replication levels in 3D41CD163 cells compared with the empty vector group, whereas no marked impact on viral replication induced by C489A was detected (Fig. 3H).
These results indicated that MALT1 degraded and downregulated N4BP1 and MCPIP1 relying on its proteolytic activity, thus facilitating PRRSV replication.
MALT1 expression is elevated upon PRRSV infection. To evaluate the impact of PRRSV infection on MALT1 expression, mRNA and protein levels of MALT1 upon PRRSV infection in Marc-145 cells were first detected using qPCR and Western blot analysis, respectively. The results showed that over a 36-h infection course, MALT1 was remarkably upregulated both in gene and protein levels (Fig. 4A). Moreover, MALT1 expression was visualized by confocal microscopy. Fluorescence signals of PRRSV N and MALT1 protein were significantly increased after PRRSV infection (Fig. 4B). Notably, merged images indicated that PRRSV N protein was colocalized with MALT1, suggesting the interaction between MALT1 and PRRSV. These results demonstrated that MALT1 could be induced by PRRSV infection.
To investigate the viral factors that induce MALT1 expression, Marc-145 cells were transfected with various plasmids expressing PRRSV nsps. The results showed that MALT1 mRNA expression was upregulated upon transfection with nsp4, -7b, and -11 (Fig. 4C). Further, MALT1 protein was significantly induced after transfection with nsp4, -7b, or -11 compared with the empty vector group, especially with nsp11 ( Fig. 4D). In addition, remarkable colocalization of nsp11 and MALT1 was observed in cells cotransfected with different nsps and MALT1 (Fig. 4E). PRRSV nsp11 has been confirmed to induce interleukin-17 (IL-17) expression, causing MCPIP1 downregulation (12). Thus, to investigate whether nsp11-induced IL-17 upregulates MALT1 expression, IL-17 recombinant protein was incubated with cells, and the results showed that IL-17 treatment markedly upregulated MALT1 protein level in dose-and time-dependent manners (Fig. 4F). On the contrary, IL-17 inhibitor Y320 dramatically suppressed nsp11-induced MALT1 expression (Fig. 4G). These results indicated that PRRSV nsp11 employed IL-17 to induce MALT1 expression. MALT1 is downregulated upon PRRSV infection aggravation. Intriguingly, 36 h after PRRSV HuN4 infection, a significant decrease in MALT1 protein level was observed in both Marc-145 cells and PAMs (Fig. 5A). Similar trends of MALT1 expression also were observed in PAMs infected with other PRRSV strains, including low-pathogenicity strain HNxx16 and highly pathogenic strain ZJnb16-2 (6, 26) (Fig. 5B). MALT1 levels  PRRSV Regulates MALT1 to Adapt Innate Immunity mBio peak occurs even before 12 hpi in PAMs. These results indicated that MALT1 is dramatically downregulated upon PRRSV infection aggravation after the initial induction of MALT1 upon PRRSV infection.
To confirm the protein degradation pathway of MALT1 after PRRSV heavy infection, proteasome inhibitor MG-132 or lysosome inhibitor chloroquine (CQ) was incubated with PRRSV-infected Marc-145 cells and PAMs. The results showed that treatment with MG-132 recovered MALT1 expression at 48 hpi, whereas CQ made no difference to the downregulation of MALT1 (Fig. 5E). On this basis, coimmunoprecipitation (co-IP) assay was performed to detect the ubiquitination level of MALT1 after PRRSV infection, and the results indicated that MALT1 was ubiquitinated after PRRSV infection compared with uninfected cells, especially at 48 hpi (Fig. 5F), indicating that MALT1 was degraded via the ubiquitin-proteasome pathway after heavy infection of PRRSV. nsp6-mediated MALT1 degradation relieves inflammatory responses. Furthermore, to identify the vital viral factor that induces MALT1 downregulation, nsps that have potential downregulation effects on MALT1 (Fig. 4C) were used to transfect Marc-145 cells, including nsp2, -3, -6, -7a, -8, -10, and -12. The results of Western blot analysis showed that among these nsps, nsp6 could markedly suppress MALT1 protein expression 24 h after transfection (Fig. 6A). To further confirm the downregulation effect of nsp6 on MALT1, cells were collected at different time points after nsp6 transfection for Western blot detection. MALT1 protein levels were found to significantly reduce from 24 h after nsp6 transfection (Fig. 6B). Moreover, MG-132 treatment in nsp6-transfected cells recovered MALT1 expression (Fig. 6C), indicating the correlation between PRRSV nsp6 and proteasome-mediated MALT1 degradation. Subsequently, to locate the key site of nsp6 responsible for MALT1 downregulation, various mutants with every two or three amino acids in WT nsp6 mutated to Ala were constructed (Fig. 6D), and Marc-145 cells were transfected with these mutants. The results showed that G1/K2A and L3/R4/ E5A mutations recovered MALT1 expression. On this basis, single-point mutation of possible active amino acid sites, including Lys 2, Leu 3, Arg 4, and Glu 5, was further performed, and the results of transfection experiments showed that K2A and L3A recovered MALT1 expression (Fig. 6E), indicating that Lys 2 and Leu 3 of nsp6 play critical roles in reducing MALT1 expression. In addition, the transcriptional levels of nsp6 and nsp11 after PRRSV infection were compared. The results showed that nsp11 levels were significantly higher than those of nsp6 at 24 hpi (Fig. 6F), indicating PRRSV manipulates MALT1 expression levels via differential expression of nsps at different stages of infection.
To further explore the effects of nsp6-induced MALT1 downregulation on the innate immune responses, transcription levels of NF-k B and related proinflammatory cytokines, including IL-1b, IL-6, and beta interferon (IFN-b), were detected after nsp6 transfection or MALT1 knockdown by siRNA. The results showed that si-MALT1-1 transfection significantly downregulated transcriptional levels of NF-k B1 (Fig. 6G), along with IL-1b, IL-6, and IFN-b (Fig. 6H), compared with the control siRNA. Consistent with this, nsp6 transfection also inhibited transcriptional levels of NF-k B1 (Fig. 6I) and cytokines (Fig. 6J), indicating that PRRSV nsp6 can suppress host inflammatory responses. In addition, to investigate the impact of nsp6 on RNase expression, gene and protein levels of N4BP1 and MCPIP1 after nsp6 transfection were detected. The results showed that nsp6 transfection had no significant stimulating effects on N4BP1 and MCPIP1 expression (Fig. 6K), leading to no reversion of antiviral activity. Together, these results indicated that PRRSV nsp6 suppressed inflammatory responses but did not recover RNase expression, which builds modulated immune circumstance for virus settlement.

DISCUSSION
Innate antiviral defense, including host ribonucleases (RNases) and inflammatory responses, serves as the indispensable and immediate barrier in hosts upon infection. Viruses need to develop strategies to evade, subvert, or even hijack these host defense mechanisms to adapt antiviral innate immunity and successfully establish infection. Previous studies have revealed that one of the mechanisms of PRRSV-induced immunosuppression is to inhibit type I interferon expression (4). In this study, PRRSV was confirmed to modulate the innate immunity through another pathway, manipulating MALT1 expression to antagonize host antiviral RNases and alleviate inflammatory responses (Fig. 7). PRRSV has a restricted tropism for monocyte/macrophage lineage in lungs and other tissues and preferentially targets porcine alveolar macrophages (PAMs) (27). Therefore, primarily isolated and cultured PAMs act as an appropriate infection model, which has the comparable soundness of a pig model with minimal animal usage, for pathogenesis research on PRRSV (28, 29) (including this study) and some other major porcine viruses, such as African swine fever virus (ASFV) (30) and  (27,32). In this study, MCPIP1 and N4BP1 were confirmed to inhibit PRRSV replication, relying on their RNase activity. Both RNases belong to the Zc3h12a-like NYN domain subfamily of endoribonucleases and harbor a similar PIN-like domain with RNase activity (10). MCPIP1 is a spectrum antiviral RNase against various viruses, like HIV-1, by degrading viral mRNA (11). It has been demonstrated that MCPIP1 inhibited PRRSV infection by targeting PRRSV mRNA and inhibiting virus-induced proinflammatory cytokines in our recent study (12). Here, it was further confirmed that Asp125 of porcine MCPIP1 is the key amino acid site for its RNase and anti-PRRSV activity. Notably, a recent study reported that like MCPIP1, N4BP1 also inhibits HIV-1 replication via degrading HIV mRNA species (13). Here, N4BP1 was confirmed to have synergistic effects with MCPIP1 on suppressing PRRSV infection. Interestingly, MCPIP1 is a cytoplasmic RNase, whereas N4BP1 mainly localizes on the nucleus and, thus, might degrade distinguished mRNAs and control different biological processes. Together, N4BP1 cooperates with MCPIP1 to suppress specific virus infection, including HIV-1 and PRRSV. Further studies on the antiviral spectrum of the two RNases and their cooperative interaction should provide additional insights into functions of host RNases.
Although N4BP1 and MCPIP1 directly degrade HIV-1 RNA in quiescent CD4 1 T cells, MALT1 caused their degradation and inactivation upon T cell receptor stimulation, reactivating HIV-1 proviruses in latently infected cells (11,13,14). Thus, MALT1 protease-mediated inactivation of the two RNases, N4BP1 and MCPIP1, facilitates the reactivation of latent  HIV-1. Similarly, in this study, MALT1 was confirmed to antagonize expression of N4BP1 and MCPIP1. Importantly, MALT1 was rapidly induced upon PRRSV infection, antagonizing the anti-PRRSV effects of the two RNases relying on its proteolytic activity and, thus, facilitating PRRSV replication. Mi-2 is a small-molecule inhibitor that covalently binds to the catalytic center of MALT1 caspase-like protease domain and inhibits its proteolytic activity (18). In this study, Mi-2 treatment caused accumulation of N4BP1 and MCPIP1 and, importantly, inhibited PRRSV replication. In addition, MALT1 protease activity against the two RNases was further verified by mutation of its caspase-like domain. Porcine MALT1 mutant with C489A was deprived of its proteolytic activity on N4BP1 and MCPIP1 and thus lost its function in promoting PRRSV replication. Together, PRRSV-induced MALT1 expression antagonizes the antiviral effects of host RNases N4BP1 and MCPIP1, relying on its proteolytic activity and, as a result, facilitating PRRSV multiplication. In view of the known and potential antiviral effects of host RNases, the significance of MALT1 protease-induced RNase degradation in pathogenesis of other viruses will be a valuable research direction in the future. In addition, PRRSV nsp4, nsp7b, and nsp11 were confirmed to induce MALT1 expression, especially nsp11. Previous studies verified that several PRRSV nsps, including nsp1a and -1b (33), nsp2 (34), nsp4 (35), and nsp11 (36,37), induce host immunosuppression via IFN inhibition. Here, PRRSV nsp-induced MALT1 upregulation may be a new mechanism for immune suppression via antagonizing host antiviral RNases. Nsp11 was recently demonstrated to induce IL-17 expression and thus antagonize the anti-PRRSV activity of MCPIP1 (12). In this study, it was further confirmed that IL-17 was responsible for nsp11induced MALT1 upregulation. Therefore, PRRSV nsp11 may rely on IL-17 to induce MALT1, consequently causing degradation of antiviral RNases like MCPIP1 and N4BP1.
Upon viral infection, various antiviral molecules, such as IFNs and other proinflammatory cytokines, are activated by recognition of pathogen-associated molecular patterns (PAMPs) through a set of pattern recognition receptors (PRRs) (38,39). Current studies have revealed that PRRSV inhibits the antiviral IFN response via encoding multiple nsps to suppress the RIG-I signaling pathway, degrade MAVS, and interfere with the phosphorylation of IRF3 and the polyubiquitination of Ik Ba (40)(41)(42)(43). Similarly, picornavirus (44), Pox viruses (45), HIV-1 (46), and severe acute respiratory syndrome coronavirus 2 (47) impair IFN response to evade host immune response. Overall, viruses need to relieve innate inflammatory responses to establish infection successfully. Intriguingly, in this study, it was found that infection aggravation of PRRSV induced the downregulation of initially elevated MALT1. MALT1 protein levels dramatically decreased at the late stage of PRRSV infection (especially after 36 hpi) with no strain specificity, and a high dose of viral infection (MOI of 10) induced more rapid decrease of MALT1. Given that MALT1 positively regulates innate immunity and inflammation, the significance of MALT1 decrease is possibly to mollify inflammatory responses induced by viral infection. As expected, in this study, MALT1 knockdown by siRNA inhibited NF-k B signals and production of proinflammatory cytokines, including IL-1b, IL-6, and IFN-b. Specifically, MALT1 facilitates NF-k B activation for proinflammatory gene transcription via both scaffolding and protease activities. On the one hand, as a scaffold protein in the CBM signaling complex, MALT1 recruits and assembles downstream effector proteins for IKK and NF-k B activation (22,48). On the other hand, the protease activity of MALT1 causes degradation of negative NF-k B regulators for NF-k B signal enforcement, including the NF-k B inhibitor protein A20 (20), deubiquitinase CYLD (19), and NF-k B member RelB (49). Overexpression of MALT1 can lead to nonstop NF-k B activation without the need for upstream signaling (50). MALT1 also cleaves an entire set of mRNA stability regulators for proinflammatory cytokine production, including Roquin-1 and Roquin-2 as well as MCPIP1 and N4BP1 (51). In addition, MALT1 proteolytic activity plays critical roles in JNK (19) and mTOR kinase pathway activation (52). Together, MALT1 acts as an activator in inducing NF-k B activation and proinflammatory cytokine production, while PRRSV-mediated MALT1 downregulation upon infection aggravation avoids aggressive inflammatory responses.
PRRSV drives MALT1 downregulation upon infection aggravation, which is in absence upon stimulation with other PAMPs, like LPS, implying the unique mechanism on MALT1 from PRRSV is involved in the induction of immune suppression. Treatment of proteasome inhibitor MG-132 and co-IP analysis confirmed that MALT1 was degraded via the ubiquitinproteasome pathway at the late infection stage, and PRRSV nsp6 could independently mediate this process. Further site analysis indicated that Lys 2 and Leu 3 of nsp6 are the critical amino acid sites responsible for MALT1 degradation. Consistent with the results of MALT1 knockdown, nsp6 transfection suppressed transcriptional levels of proinflammatory genes, including IL-1b, IL-6, and IFN-b. These cytokines play key roles in the defense against virus infection. Importantly, nsp6 had no direct effects on expression of anti-PRRSV RNases, including N4BP1 and MCPIP1. Therefore, PRRSV nsp6-mediated MALT1 degradation may be a novel mechanism for PRRSV-induced innate immune suppression. The function of PRRSV nsp6 is largely unknown. One group reported that PRRSV nsp5, nsp6, and nsp7, along with their orthologous nsp6 proteins of several coronaviruses, including infectious bronchitis virus, mouse hepatitis virus, and severe acute respiratory syndrome, can induce small-diameter autophagosomes, activating autophagy (53,54). Together with the role of nsp6 in proteasome-mediated degradation of MALT1 identified in this study, PRRSV nsp6 seems to act as an activator in the cellular protein degradation system. Together, PRRSV nsps may accurately modulate MALT1 expression at different infection states. PRRSV employs nsps, including nsp4, -7b, and -11, to upregulate MALT1 and antagonize RNase activity, facilitating PRRSV initial replication, and MALT1 was downregulated by PRRSV nsp6 to suppress virus-induced excessive inflammatory responses and rebuild homeostasis in infected host immunity.
In addition, with the discovery of MALT1 proteolytic activity, research and treatment targeting MALT1 proteolytic activity have been reported. MALT1 protease-dead (MALT1 PD) in mice significantly changed the immunophenotype. MALT1 PD mice develop serious autoimmunity with inflammation and lymphocyte infiltration in multiple organs (22,55), suggesting the role of MALT1 protease activity in immune homeostasis. Notably, regulatory T cell (Treg) numbers are reduced in MALT1 PD mice (22,56), whereas Th1 and T helper (Th2) cells are increased (55), implying the significance of MALT1 protease activity in T cell activation. Whether MALT1-induced Treg activation contributes to immunosuppression caused by PRRSV deserves further study. In addition, MALT1 protease inhibitor Mi-2 recovers MCPIP1 expression, selectively reducing HIV-1 latently infected CD4 1 T cells (57). Therefore, MALT1 protease has potential as a therapeutic target against PRRSV infection.
To sum up, PRRSV induced MALT1 protease to antagonize anti-PRRSV effects of the two RNases MCPIP1 and N4BP1, facilitating PRRSV replication. Although multiple PRRSV nsps induced MALT1 expression, nsp6 mediated MALT1 degradation via the ubiquitination-proteasome pathway, indicating elaborate regulatory effects of PRRSV on MALT1 expression. Considering the potential risk in immune pathogenesis caused by excessive MALT1, nsp6-mediated MALT1 degradation may contribute to immune alleviation, which is a benefit for long-term virus infection. For the first time, these findings revealed the precise regulatory machinery of PRRSV on MALT1 for both the antagonistic effects against the host RNases and the recovery of homeostasis in immune system postinfection, enlightening the new mechanism of PRRSV for successful immune suppression and virus survival by manipulating MALT1 expression.
Virus infection. Cells were grown to approximately 70% to 80% confluence and infected with viruses. Infection was allowed to proceed at 37°C and 5% CO 2 for 2 h, and supernatants were then removed. Cell monolayers were rinsed with Hanks' balanced salt solution to remove unattached virus particles and then incubated in the presence of fresh medium containing 2% FBS at 37°C and 5% CO 2 for designated time periods to investigate effects of virus infection on target proteins.
RNA interference. Small interference RNAs (siRNAs) against monkey or pig MALT1 and siRNA-negative control (NC) were designed and synthesized by GenePharma (Shanghai, China). Marc-145 and 3D4 cells were transfected with the indicated siRNAs at a final concentration of 50 nM using Lipofectamine 2000 (Thermo) according to the manufacturer's instructions for 48 h. Sequences of siRNAs were listed in Table 2.
RNA isolation and qRT-PCR. Total RNA was extracted using the RNA extraction kit (Easy-Do, Zhejiang, China) according to the manufacturer's instruction and reverse transcribed to cDNA using HiScript III 1st strand cDNA synthesis kit (1gDNA wiper) (Vazyme, Nanjing, China). qRT-PCR was performed using SYBR green PCR mix (Vazyme) on a Stratagene Mx3005P real-time PCR system (Agilent Technologies, Santa Clara, CA, USA). The PCR program included one denaturation cycle at 95°C for 30 s, followed by 40 amplification cycles at 95°C for 5 s and 60°C for 45 s. One final melting cycle to produce a melting curve was added to verify product specificity, and a single peak obtained in the melting curve indicated the specificity of the PCR products. The relative gene expression levels were normalized to the housekeeping gene b-actin or GAPDH. The 2 2DDCt method was used to determine the number of fold change in gene expression levels. All primers for qRT-PCR were listed in Table 3.
Co-IP. Marc-145 cells were lysed using radioimmunoprecipitation assay lysis buffer (RIPA) (Beyotime, Shanghai, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime). Immunopre-cipitation was performed using protein A1G agarose (Beyotime) according to the manufacturer's instructions. Briefly, protein samples were first incubated with anti-MALT1 antibodies (1:50 diluted) at 4°C overnight, followed by incubation with 40 mL of protein A1G agarose at 4°C for 2 h. Agarose-Ab-antigen complexes were washed with ice-cold phosphate-buffered saline (PBS) five times and then resuspended with 40 mL 1ÂSDS- PAGE buffer. Precipitates and whole-cell lysates were subjected to 12% SDS-PAGE, and MALT1 ubiquitination was analyzed by Western blotting using ubiquitin rabbit pAb (1:500; Beyotime). Western blotting. Briefly, cell or tissue samples were lysed using RIPA (Beyotime) to extract total proteins. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime) and 50 mg of protein was separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Billerica, MA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline-Tween 20 (TBST) for 1 h at room temperature. Rinsed blots were then incubated with primary antibody overnight at 4°C, followed by HRP-conjugated goat anti-mouse or antirabbit IgG (1:5,000) at 37°C for 1 h. Signals were detected with SuperSignal West Pico/Femto chemiluminescent substrate (Thermo Scientific, MA, USA), and images were captured with a Gel 3100 chemiluminescent imaging system (Sage Creation Science, Beijing, China).
Indirect immunofluorescence assays and confocal microscopy. Briefly, cells in culture plates (Costar; Corning, NY, USA) were first fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 15 min. Fixed cells were washed with PBS and blocked with 5% goat serum in PBS for 1 h at 37°C to prevent nonspecific binding. Cells were incubated with corresponding primary antibodies at 37°C for 1 h and stained with Alexa Fluor-conjugated IgG (H1L) (1:1,000) in the dark at 37°C for 1 h. Cellular nuclei were counterstained with nuclear dye 49,6-diamidino-2-phenylindole (DAPI) (1:2,000 diluted in PBS; Beyotime) for 5 min. Cells were observed under either inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) or confocal laser scanning  Statistical analysis. Data were obtained from at least three independent experiments for the quantitative analysis and were expressed as means 6 standard errors of the means. All statistical analyses were performed with t test or one-way analysis of variance (ANOVA). A P value of ,0.05 was considered a significant difference.