Porcine Reproductive and Respiratory Syndrome Virus Nonstructural

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Cleaves Porcine DCP1a To Attenuate Its Antiviral Activity This information is current as of September 25, 2021. Ran Tao, Liurong Fang, Dongcheng Bai, Wenting Ke, Yanrong Zhou, Dang Wang and Shaobo Xiao J Immunol published online 29 August 2018 http://www.jimmunol.org/content/early/2018/08/28/jimmun ol.1701773 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 29, 2018, doi:10.4049/jimmunol.1701773 The Journal of Immunology

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Cleaves Porcine DCP1a To Attenuate Its Antiviral Activity

Ran Tao,*,† Liurong Fang,*,† Dongcheng Bai,*,† Wenting Ke,*,† Yanrong Zhou,*,† Dang Wang,*,† and Shaobo Xiao*,†

As one of the most signiﬁcant etiological agents in pigs, porcine reproductive and respiratory syndrome virus (PRRSV) has ad- versely impacted the global swine industry since it was discovered in the 1980s. The mRNA-decapping enzyme 1a (DCP1a), a reg- ulatory factor involved in removing the 59-methylguanosine cap from eukaryotic mRNA, has recently been identiﬁed as an IFN-stimulated gene. However, the role of DCP1a in PRRSV infection is not well understood. In this study, overexpression and

knockdown of porcine DCP1a (pDCP1a) showed that pDCP1a affected PRRSV infection. Interestingly, we found that PRRSV Downloaded from infection signiﬁcantly downregulated pDCP1a expression at the protein level by cleaving pDCP1a. Furthermore, we demonstrated that PRRSV nonstructural protein 4 (nsp4), a 3C-like proteinase, is responsible for pDCP1a cleavage, and the cleaved site is at glutamic acid 238 (E238) of pDCP1a. The mutant pDCP1a-E238A, which cannot be cleaved by nsp4, showed higher anti-PRRSV

activity, and the antiviral effects of two cleavage products (pDCP1a1–238 and pDCP1a239–580) were signiﬁcantly decreased compared with wild type pDCP1a. Unexpectedly, PRRSV infection or overexpression of nsp4 did not cleave monkey DCP1a, and

monkey DCP1a showed a higher anti-PRRSV activity than pDCP1a. Taken together, this study reveals a new strategy evolved by http://www.jimmunol.org/ PRRSV to dampen the host defense, complementing the known PRRSV-mediated immune evasion mechanisms. The Journal of Immunology, 2018, 201: 000–000.

orcine reproductive and respiratory syndrome virus Until now, current vaccination strategies and antiviral drugs have (PRRSV) is the causative agent of porcine reproductive not been sufﬁcient to control PRRSV infection (10–13). Thus, a P and respiratory syndrome, a highly contagious viral dis- better understanding of PRRSV-mediated immune evasion mecha- ease of pigs leading to enormous economic loss in the worldwide nisms will facilitate development of more effective control measures. swine industry (1, 2). PRRSV possesses a single-stranded, positive-

As is commonly known, the innate immune system provides the by guest on September 25, 2021 sense RNA genome of ∼15 kb and belongs to the family first line of defense against invading viruses. Initially, virus in- Arteriviridae in the order Nidovirales (3, 4). Its genome consists fection is recognized by specific host pattern recognition receptors, of at least 11 open reading frames that encode two large poly- which results in activation of the type I IFN production pathway proteins (pp1a and pp1ab) and eight structural proteins (5–7). (14). After binding their cognate receptors, type I IFNs activate Upon PRRSV infection, pp1a and pp1ab are processed into at least the JAK-STAT signaling pathway, inducing transcription of hun- 15 nonstructural proteins (nsps) by the papain-like protease nsp2 dreds of IFN-stimulated genes (ISGs) (15, 16). ISGs directly and the 3C-like protease nonstructural protein 4 (nsp4) (6, 8, 9). target multiple stages of the viral life cycle to resist viral infection (17–19). However, during coevolution of viruses and hosts, viru- *State Key Laboratory of Agricultural Microbiology, College of Veterinary Medi- ses have developed many mechanisms to counteract the antiviral † cine, Huazhong Agricultural University, Wuhan 430070, China; and Key Laboratory functions of ISGs. For instance, many kinds of viruses, including of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China PRRSV, downregulate the transcription levels of many ISGs by ORCID: 0000-0003-0023-9188 (S.X.). inhibiting type I IFN signaling (20–26). Furthermore, viral an- Received for publication December 21, 2017. Accepted for publication August 7, tagonists can cause the mislocalization of ISGs to downregulate 2018. their antiviral functions. The lentiviral Vpu protein employs this This work was supported by the National Natural Science Foundation of China strategy to antagonize tetherin (27). Some viruses can also (31490602), the National Basic Research Program (973) of China (2014CB542700), downregulate the protein levels of ISGs to suppress their antiviral and the Key Technologies R&D Program of China (2015BAD12B02). activity [for example, the Vpx protein of HIV-2 and related SIV Address correspondence and reprint requests to Prof. Shaobo Xiao, Laboratory of Animal Virology, College of Veterinary Medicine, Huazhong Agricultural University, target SAMHD1 for proteasomal degradation (28)]. A similar 1 Shi-zi-shan Street, Wuhan 430070, Hubei, People’s Republic of China. E-mail mechanism is used by the lentiviral Vif protein to antagonize address: [email protected] APOBEC3G (29, 30). Additionally, to effectively replicate in the Abbreviations used in this article: DCP1a, mRNA-decapping enzyme 1a; gRNA, presence of ISGs, viruses might also exploit other mechanisms guide RNA; HA, hemagglutinin; hDCP1a, human DCP1a; IPS-1, IFN-b promoter stimulator 1; ISG, IFN-stimulated gene; 3-MA, 3-methyladenine; mDCP1a, monkey that have not yet been characterized. DCP1a; MOI, multiplicity of infection; N, nucleocapsid; NEMO, NF-kB essential The mRNA-decapping enzyme 1a (DCP1a) is a cofactor par- modulator; nsp4, nonstructural protein 4; PAM, porcine alveolar macrophage; 9 pDCP1a, porcine DCP1a; PRRSV, porcine reproductive and respiratory syndrome ticipating in removing the 5 -methylguanosine cap from eukaryotic virus; qRT-PCR, quantitative real-time PCR; si-Ctrl, negative control siRNA; mRNA (31, 32). Recent studies showed that DCP1a is also an ISG si-pDCP1a, pDCP1a-specific siRNA1; siRNA, small interfering RNA; TCID50, induced by IFN-a (33–35). Additionally, DCP1a can restrict polio- 50% tissue culture infective dose; TD, trimerization domain. virus infection through inducing translational arrest, which is not Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 related to the localization of DCP1a to processing bodies, where it

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701773 2 PRRSV nsp4 CLEAVES PORCINE DCP1a performs its decapping function (36). However, the role of DCP1a DAPI (Beyotime) for 10 min. Fluorescent images were taken with an in PRRSV infection remains unknown. Olympus IX73 inverted microscope (Olympus). In the current study, we found that porcine DCP1a (pDCP1a) is 50% tissue culture infective dose assay for determination of an ISG and inhibits PRRSV replication. However, PRRSV infec- PRRSV titers tion could cleave pDCP1a to downregulate pDCP1a expression. We further investigated the underlying mechanism and biological PRRSV titers were expressed as 50% tissue culture infective dose (TCID50)/ml using the Reed–Muench method. Briefly, MARC-145 cells or significance of pDCP1a cleavage by PRRSV infection. We found PK-15CD163 cells were seeded in 96-well plates, then PRRSV samples that PRRSV nsp4 cleaved pDCP1a to attenuate its antiviral activity, were 10-fold serially diluted, and 100 ml was added to each well in eight revealing a new strategy evolved by PRRSV to counteract antiviral replicates. The plates were incubated for 72–96 h before virus titers were innate immunity. calculated. Western blot analysis Materials and Methods Cells cultured in 60-mm dishes were harvested with lysis buffer (Beyotime). Cells and virus The cell extracts were then resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Protein expression was HEK-293T, MARC-145 cells (a monkey kidney cell line), and PK-15CD163 analyzed using appropriate specific Abs. The expression of b-actin was cells (a porcine kidney cell line expressing PRRSV receptor CD163, gifted detected to demonstrate equal protein sample loading throughout from Dr. E.-m. Zhou at Northwest A&F University, China) were cultured this study. in DMEM (Invitrogen) supplemented with 10% FBS at 37˚C in a hu- Generation of CRISPR/Cas9–mediated pDCP1a knockout cells midified atmosphere with 5% CO2. Porcine alveolar macrophages (PAMs) Downloaded from were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml CD163 Knockout of the pDCP1a gene in PK-15 cells was performed by m penicillin, and 100 g/ml streptomycin. The highly pathogenic type 2 using the CRISPR/Cas9 system. Briefly, two guide RNAs (gRNAs) tar- PRRSV strain WUH3 (GenBank accession no. HM853673), which was geting pDCP1a (gRNA1: 59-CGGCCA TGAGTTTGGCTATC-39; gRNA2: isolated from pigs showing signs of “high fever syndrome” at the end of 59-TCACCGGATAGCCAAACTCA-39) were synthesized using the GeneArt 2006 (37), was used throughout this study. PRRSV was propagated in CD163 Precision gRNA Synthesis Kit (Invitrogen) according to the manufacturer’s MARC-145 or PK-15 cells. CD163 instructions. PK-15 cells were transiently transfected with gRNA1 or Reagents and Abs gRNA2 along with the Cas9 protein (Invitrogen). After 72-h incubation, cells were treated with trypsin and harvested to estimate the gene editing http://www.jimmunol.org/ A proteasome inhibitor (MG132) and autophagy inhibitor (3-methyladenine efficiency. Single-cell clones were generated using limiting dilution. [3-MA]), purchased from Sigma-Aldrich (St Louis, MO), were used at pDCP1a gene knockout was confirmed by genomic DNA sequence and concentrations of 10 mM and 5 mM, respectively. A pan-caspase inhibitor Western blot analysis. (Z-VAD-FMK; Beyotime, Jiangsu, China) was used at a concentration of 20 mM. Mouse mAbs against FLAG and hemagglutinin (HA) were pur- Statistical analyses chased from Medical & Biological Laboratories (Nagoya, Japan). Anti–b- actin Ab was obtained from Antgene (Wuhan, China). Rabbit polyclonal Abs The results were analyzed for significance by Student t test using GraphPad against DCP1a were purchased from Abcam (Cambridge, U.K.). Mouse Prism 6 software. Differences between groups were considered statistically , mAbs against PRRSV nucleocapsid (N) proteins were made in-house (38). significant when the p values were 0.05. All experiments were repeated at least three times. Plasmids and small interfering RNAs by guest on September 25, 2021 The pDCP1a and monkey DCP1a (mDCP1a) expression plasmids were CD163 Results constructed by RT-PCR amplification from cDNA of PK-15 cells or pDCP1a is an ISG, and overexpression of pDCP1a slightly MARC-145 cells, respectively, and cloned into a pCAGGS-Flag vector with an N-terminal Flag tag. All pDCP1a substitution and deletion mutants inhibits PRRSV replication were also cloned into pCAGGS-Flag. The PRRSV nsp4 gene was cloned Previous studies showed that human DCP1a (hDCP1a) is an ISG into a pCAGGS-HA vector with a C-terminal HA tag. PRRSV nsp4 mutants were also inserted into the pCAGGS-HA vector. All constructs were induced by IFN-a (33). To determine whether pDCP1a can be confirmed by DNA sequencing. Small interfering RNAs (siRNAs) target- induced by IFNs in PAMs, this cell type was treated with porcine ing pDCP1a or negative control siRNA (Invitrogen) were transfected at a IFN-a at a concentration of 1000 U/ml, followed by detection of working concentration of 50 nM. The sequences of siRNAs and primers mRNA levels of pDCP1a at different time points after treatment. for plasmid construction can be provided upon request. In this study, the mRNA level of ISG15 was also detected in RNA extraction and quantitative real-time PCR parallel from the same samples. The results showed that both pDCP1a and ISG15 could be induced by IFN-a in PAMs (Fig. 1A, Total RNA from cultured cells was extracted using TRIzol reagent (Omega Bio-Tek, Norcross, GA). RNAwas then reverse transcribed to cDNA using a 1B). However, the fold induction of pDCP1a was lower than Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, that of ISG15. These results demonstrate that pDCP1a is an ISG Germany) according to the manufacturer’s instructions. Quantitative real- induced by IFN-a in PAMs. time PCR (qRT-PCR) was performed with SYBR Green Real-Time PCR Because pDCP1a is an ISG, we then investigated the effect of Master Mixes (Applied Biosystems, Foster City, CA) in the Applied CD163 Biosystems ViiA 7 Real-Time PCR System (Life Technologies). The pDCP1a on PRRSV replication. PK-15 cells were transiently mRNA levels of target genes were relatively quantified using the 22DDCt transfected with pCAGGS-pDCP1a-Flag or an empty vector and method. The GAPDH gene served as an internal reference. Genomic RNA then infected with PRRSV strain WUH3 at an MOI of 0.5. As copies of PRRSV were quantified by detecting the PRRSV nsp9 gene as shown in Fig. 1C and 1D, both qRT-PCR and TCID50 assays described previously (39). showed that overexpression of pDCP1a slightly suppressed Indirect immunofluorescence assay PRRSV replication at different time points (12, 24, and 36 h) after PRRSV infection. PK-15CD163 cells were seeded on circular glass coverslips in 24-well plates and then infected with PRRSV at a multiplicity of infection (MOI) of 0.5. We further investigated whether the IFN-induced gene products At the indicated time points postinfection, cells were fixed with 4% could cooperate with pDCP1a to potentiate the antiviral activity. paraformaldehyde for 15 min and then permeabilized with precooled To this end, PK-15CD163 cells were pretreated with IFN-a for 12 h methanol for 10 min. After blocking with 5% BSA for 1 h, cells were and then transfected with pCAGGS-pDCP1a-Flag or an empty vec- incubated with primary Abs (mAbs against PRRSV N protein and rabbit polyclonal Abs against DCP1a) for 1 h at room temperature. Cells were tor. At 36 h posttransfection, cells were challenged with PRRSV then incubated with Alexa Fluor 594–conjugated donkey anti-mouse or (MOI = 0.5). As shown in Fig. 1E and 1F, overexpression of pDCP1a 647-conjugated donkey anti-rabbit Abs (Antgene) for 45 min, followed by in IFN-a–pretreated PK-15CD163 cells enhanced IFN-a–mediated The Journal of Immunology 3

FIGURE 1. pDCP1a is an ISG, and overexpression of pDCP1a slightly inhibits PRRSV replication. (A and B) PAMs were treated with porcine IFN-a (1000 U/ml) for 4, 8, and 12 h, and then cells were harvested to analyze the expression of pDCP1a and ISG15 by qRT-PCR. (C and D) PK-15CD163 cells were transfected with pDCP1a expression plasmid or control vector. At 24 h posttransfection, cells were in-

fected with PRRSV strain WUH3 for different Downloaded from times (12, 24, and 36 h) at an MOI of 0.5. Cells were then harvested for qRT-PCR analysis (C) CD163 or TCID50 assays (D). (E and F) PK-15 cells were pretreated with IFN-a (1000 U/ml) for 12 h and then transfected with pDCP1a expression plasmid or control vector. At 24 h posttransfection, cells were infected with PRRSV http://www.jimmunol.org/ (MOI = 0.5) for 36 h. Cells were collected for qRT-PCR analysis (E) or TCID50 assays (F). Results from three independent experiments (mean 6 SD) are shown. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 25, 2021

antiviral effects, suggesting that IFN-inducible products cooperate PK-15CD163 cells reversed the IFN-a–mediated inhibition of PRRSV with pDCP1a to potentiate the antiviral action. replication. Similar effects were also observed in PAMs (Fig. 2H, 2I). These results demonstrate that knockdown of pDCP1a could diminish Knockdown of pDCP1a by siRNA promotes PRRSV replication IFN-a’s anti-PRRSV effects in both PK-15CD163 cells and PAMs. To further validate the effect of pDCP1a overexpression on PRRSV replication, we tested whether knockdown of pDCP1a could PRRSV infection cleaves pDCP1a promote PRRSV replication. We designed three siRNAs targeting To further investigate whether PRRSV infection affects pDCP1a pDCP1a and examined their interference effects in PK-15CD163 expression, we evaluated the mRNA levels and subcellular lo- cells by qRT-PCR and Western blot analysis. siRNA1 showed the calization of pDCP1a in PK-15CD163 cells infected with PRRSV at highest knockdown efficiency and was selected for subsequent different time points. As displayed in Fig. 3A and 3B, PRRSV experiments (Fig. 2A). PK-15CD163 cells were transiently trans- infection did not affect the mRNA levels of pDCP1a, nor did it fected with siRNA1 (pDCP1a-specific siRNA1 [si-pDCP1a]) have an impact on the subcellular localization of pDCP1a. We or negative control siRNA (si-Ctrl), respectively. At 30 h post- next analyzed the protein levels of pDCP1a in PK-15CD163 cells transfection, cells were infected with PRRSV for 24 h. As upon PRRSV infection. To our surprise, PRRSV infection sig- expected, knockdown of pDCP1a in PK-15CD163 cells increased nificantly downregulated pDCP1a protein expression. In addition, PRRSV replication (Fig. 2B, 2C). As shown in Fig. 2D and 2E, a small cleavage band of pDCP1a was observed by Western blot similar results were observed in PAMs. Together, these data in- analysis during PRRSV infection (Fig. 3C). These results indicate dicate that pDCP1a is involved in restricting PRRSV replication. that PRRSV downregulates pDCP1a expression by cleavage. We further investigated whether silencing pDCP1a could rescue PRRSV replication in IFN-a–pretreated cells. IFN-a– PRRSV nsp4 cleaves pDCP1a depending on its pretreated PK-15CD163 cells or PAMs were transfected with si-Ctrl or protease activity si-pDCP1a, respectively. At 30 h posttransfection, cells were infected To identify which PRRSV-encoded protein is responsible for with PRRSV. As shown in Fig. 2F and 2G, knockdown of pDCP1a in pDCP1a cleavage, we screened all nonstructural and structural 4 PRRSV nsp4 CLEAVES PORCINE DCP1a Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Knockdown of pDCP1a by siRNA promotes PRRSV replication. (A)PK-15CD163 cells were transfected with pDCP1a-speciﬁc siRNAs or negative control siRNA (NC) for 30 h. Cells were then harvested to examine the knockdown efﬁciency by qRT-PCR and Western blot analysis (A). (B and C) PK-15CD163 cells were transfected with si-pDCP1a or si-Ctrl for 30 h and then infected with PRRSV at an MOI of 0.5 for 24 h. Cells were collected to analyze

PRRSV RNA by qRT-PCR (B) and virus titers by TCID50 assay (C). (D and E) PAMs were transiently transfected with si-DCP1a or si-Ctrl for 30 h followed by CD163 PRRSV infection. At 24 h postinfection, viral replication was determined by qRT-PCR (D)andTCID50 assay (E). (F and G)PK-15 cells were pretreated with IFN-a (1000 U/ml) for 12 h and then transfected with si-Ctrl or si-pDCP1a. At 30 h after transfection, cells were challenged with PRRSV (MOI = 0.5) for

24 h. Cells were collected to analyze PRRSV RNA by qRT-PCR (F) and virus titers by TCID50 assay (G). (H and I)IFN-a–pretreated PAMs were transiently transfected with si-Ctrl or si-pDCP1a. At 30 h posttransfection, cells were infected with PRRSV (MOI = 0.5). Cells were harvested to determine PRRSV

RNA by qRT-PCR (H) and virus titers by TCID50 assay (I). Data were obtained from three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. proteins encoded by PRRSV. HEK-293T cells were cotransfected catalytic triad, and any mutation to these amino acids will abolish with pCAGGS-pDCP1a-Flag and expression constructs encoding the the protease activity of PRRSV nsp4 (43). To test whether nsp4 nonstructural and structural proteins of PRRSV (40), followed by protease activity is required for pDCP1 cleavage, we constructed Western blot analyses. A small cleavage band of pDCP1a, which is three catalytically deﬁcient mutants: nsp4-H39A, nsp4-D64A, and identical to the cleaved pDCP1a in PRRSV-infected cells (Fig. 3C), nsp4-S118A. As shown in Fig. 4C, pDCP1a cleavage was not could only be observed in cells overexpressing nsp4 (data not observed in cells cotransfected with pCAGGS-pDCP1a-Flag and shown), and this cleavage was dose dependent (Fig. 4A). Further- expression constructs of nsp4-H39A, nsp4-D64A, or nsp4-S118A. more, the cleavage of pDCP1a by PRRSV nsp4 was not affected by These ﬁndings suggest that PRRSV nsp4–mediated cleavage of treatment with the proteasome inhibitor MG132, the caspase in- pDCP1a depends on the protease activity of nsp4. hibitor Z-VAD-FMK, or the autophagy inhibitor 3-MA (Fig. 4B), indicating that nsp4-mediated cleavage occurred independent of PRRSV nsp4 cleaves pDCP1a at E238 cellular caspases, proteasome pathways, and autophagy pathways. We next investigated the sequence of pDCP1a for potential PRRSV PRRSV nsp4 is a 3C-like protease responsible for most cleav- nsp4 cleavage sites. Previous studies showed that a preference for ages within the viral polyprotein (41, 42). Residues His39 (H39), substrate cleavage by PRRSV nsp4 is a Glu (E) residue at the P1 Asp64 (D64), and Ser118 (S118) of PRRSV nsp4 form a canonical position (Fig. 5A) (42). As pDCP1a cleavage produced an The Journal of Immunology 5

FIGURE 3. PRRSV infection cleaves pDCP1a. (A)PK-15CD163 cells were infected with PPRSV at an MOI of 0.5 for 12, 24, and 36 h. Cells were collected to detect pDCP1a mRNA expression by qRT-PCR. Results were obtained from three independent experiments (mean 6 SD). N.S., not significant. (B) PK-15CD163 cells were infected with PRRSV at an MOI of 0.5. At 12, 24, and 36 h postinfection (hpi), cells were fixed and then stained with anti-PRRSV N protein Ab and anti-DCP1a Ab, followed by incubation with Alexa Fluor 594–conjugated donkey anti- mouse Abs for PRRSV N protein (green) and 647-conjugated donkey anti-rabbit Abs for DCP1a (red). Nuclei were stained with DAPI (blue). Original magnification 340; insets 32. Downloaded from (C) PK-15CD163 cells were challenged with PRRSV (MOI = 0.5) for the indicated times, and cell lysates were used for Western blot analysis to detect pDCP1a and PRRSV N protein. b-Actin was used as a loading control. Image J software was used to analyze the rel- ative levels of pDCP1a in comparison with http://www.jimmunol.org/ mock-infected cells, and the ratio was displayed as fold change below the images.

∼35-kDa (N-terminal) product, we hypothesized that the recog- We further evaluated the antiviral effects of different pDCP1a by guest on September 25, 2021 nized E residue in the P1 position may exist between aa 230 and mutants in pDCP1a-deficient cell lines, which were established by 243 (Fig. 5B). To test this, five E residues (E230, E238, E240, using the CRISPR/Cas9 system. Two gRNAs were designed to E241, and E243) were subsequently replaced with Ala (A) target exon 4 of the pDCP1a gene in PK-15CD163 cells. The iso- (Fig. 5B) and transfected into HEK-293T cells along with PRRSV lated PK-15CD163-DCP1aKO cell clones were confirmed by geno- nsp4. As shown in Fig. 5C, the cleaved products disappeared mic DNA sequencing and Western blot analyses (Fig. 6C, 6D). We under the expression of the E238A mutation, whereas other mu- selected one of the knockout cell lines to perform sequencing tants were still cleaved by PRRSV nsp4, indicating that E238 of analysis, which showed a 14-nt deletion and mutation in exon 4 of pDCP1a is the cleavage site targeted by PRRSV nsp4. the pDCP1a genome (Fig. 6C). The nucleotide deletion and mutation resulted in producing a nonfunctioning truncated peptide PRRSV nsp4–mediated cleavage impairs the antiviral activity (Fig. 6C). Thus, this pDCP1a-deficient cell line was used in the of pDCP1a subsequent experiments. pDCP1a and its individual mutants were As PRRSV nsp4 was found to cleave pDCP1a at E238, two transiently transfected into PK-15CD163-DCP1aKO cells, respec- deletion mutants (pDCP1a1–238 [N-terminal deletion mutant] tively. At 24 h posttransfection, cells were challenged with and pDCP1a239–580 [C-terminal deletion mutant]) encoding PRRSV for 24 h. As shown in Fig. 6E and 6F, the pDCP1a-E238A pDCP1a cleavage products were constructed. Then, pDCP1a, mutant showed a higher antiviral activity than the wild type pDCP1a-E238A, or individual deletion mutants were tran- pDCP1a, the pDCP1a1–238 mutant exhibited partial inhibitory siently transfected into MARC-145 cells followed by PRRSV ability, and the pDCP1a239–580 mutant completely abolished an- infection for 24 h. qRT-PCR and TCID50 assays were used to tiviral activity. These results from pDCP1a knockout cell lines analyze the disparities in PRRSV replication between pDCP1a further demonstrated that PRRSV nsp4–mediated cleavage im- and individual pDCP1a mutants. As shown in Fig. 6A and 6B, pairs the antiviral activity of pDCP1a. the pDCP1a-E238A mutant showed a higher antiviral activity against PRRSV compared with pDCP1a. As expected, com- PRRSV nsp4 does not cleave mDCP1a pared with pDCP1a, the inhibitory effects of the two deletion By comparing the amino acid sequences of mDCP1a, hDCP1a, and mutants, pDCP1a1–238 and pDCP1a239–580, against PRRSV pDCP1a, we found that the corresponding amino acid at residue were significantly decreased (Fig. 6A, 6B). In addition, the 238 of mDCP1a and hDCP1a was Asp (D), which was different pDCP1a239–580 mutant completely abolished antiviral activity, from that of pDCP1a (Fig. 7A). MARC-145 cells are derived from whereas the pDCP1a1–238 mutant maintained partial inhibitory monkey kidneys and used extensively for PRRSV study (44). We ability. Taken together, these data demonstrate that PRRSV then hypothesized that PRRSV nsp4 probably does not cleave nsp4–mediated cleavage impairs the antiviral activity of mDCP1a. To verify this hypothesis, we evaluated the protein pDCP1a. levels of mDCP1a in MARC-145 cells infected with PRRSV at 6 PRRSV nsp4 CLEAVES PORCINE DCP1a

FIGURE 4. PRRSV nsp4 cleaves pDCP1a depending on its protease activity. (A) HEK-293T cells were cotransfected with Flag-tagged pDCP1a and increasing doses (0.1, 0.2, 0.4 mg) of PRRSV nsp4 expression plasmids. At 28 h posttransfection, Western blotting was performed to detect Flag-tagged Downloaded from pDCP1a and HA-tagged PRRSV nsp4. (B) HEK-293T cells were transfected with Flag-tagged pDCP1a and HA-tagged PRRSV nsp4 expression plasmids. At 18 h posttransfection, cells were treated with MG132 (10 mM), Z-VAD-FMK (20 mM), or 3-MA (5 mM) for 10 h. Cell lysates were analyzed by Western blotting. (C) HEK-293T cells were transfected with Flag-tagged pDCP1a along with the wild type PRRSV nsp4 or its protease-defective mutant (nsp4- H39A, nsp4-D64A, and nsp4-S118A) expression plasmids. Cells were collected and lysed for Western blot analysis at 30 h after transfection. different time points or transfected with PRRSV nsp4. As shown transcription (21, 50, 51), 2) altering the normal subcellular lo- http://www.jimmunol.org/ in Fig. 7B and 7C, PRRSV infection had no effect on mDCP1a calization of ISGs and thus downregulating their functional per- protein expression, and PRRSV nsp4 did not cleave mDCP1a. formance (52), and 3) downregulating ISG expression by targeting Based on the above results, we examined whether mDCP1a the products to proteasomal pathways (52). For example, PRRSV showed higher antiviral activity against PRRSV compared with inhibits type I IFN signaling by blocking STAT1/STAT2 nuclear pDCP1a. Expression constructs containing mDCP1a and pDCP1a translocation, leading to downregulation of the mRNA levels of were transiently transfected into MARC-145 cells followed by ISGs (51); PRRSV E protein sequesters tetherin away from the PRRSV infection. Both qRT-PCR and TCID50 assays indicated cell surface to the cytoplasm to disrupt its antiviral activity (52); that mDCP1a showed a higher inhibitory activity against PRRSV PRRSV nsp3 degrades IFITM1 in a proteasome-dependent man-

than pDCP1a (Fig. 7D, 7E). ner to promote PRRSV infection (52). In this study, we found that by guest on September 25, 2021 PRRSV infection had no obvious effects on the mRNA levels and Discussion subcellular localization of pDCP1a; however, further investigations It is well known that IFNs have been shown to be the most crucial demonstrated that pDCP1 was directly cleaved after PRRSV infec- innate antiviral cytokines since their discovery. IFNs exert antiviral tion,revealinganewstrategyemployedbyPRRSVtoattenuatethe effects through inducing the expression of hundreds of ISGs antiviral activity of ISGs. (34, 35). However, during coevolution with hosts, viruses have As a major protease encoded by PRRSV, nsp4 plays an important developed multiple mechanisms to escape host immune surveil- role in subverting host innate immune responses (53). Previous lance by modulating innate immunity, including ISGs (20, 45–48). studies showed that PRRSV nsp4 antagonizes type I IFN pro- Similar to many other viruses, PRRSV can directly or indirectly duction by cleaving adaptor IFN-b promoter stimulator 1 (IPS-1, target ISGs to impair host antiviral responses except inhibiting also known as MAVS/VISA/Cardiff) and kinase NF-kB essential type I IFN production (49). Previous studies showed that PRRSV modulator (NEMO, also called IKKg) (54–56). However, whether counteracts ISGs by three major mechanisms: 1) inhibiting ISG PRRSV nsp4 targets ISGs has not been reported. In this study, we

FIGURE 5. PRRSV nsp4 cleaves pDCP1a at E238. (A) Sequence logo of PRRSV nsp4 cleaving the polyprotein junctions. An amino acid sequence logo of the substrate was generated by WebLogo, version 3 (http://weblogo.threeplusone.com/). (B) Schematic representation of pDCP1a and its mutants. (C) HEK-239T cells were transfected with Flag-tagged WT pDCP1a or pDCP1a mutants as indicated along with PRRSV nsp4. At 30 h posttransfection, cell lysates were prepared and analyzed by Western blotting. WT, wild type. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. PRRSV nsp4–mediated cleavage impairs the antiviral activity of pDCP1a. (A and B) pDCP1a and its mutants were transfected into MARC- 145 cells as indicated. At 24 h posttransfection, cells were challenged with PRRSV (MOI = 0.5). Cells were harvested to determine PRRSV RNA by qRT-PCR (A) or PRRSV titers by TCID50 assay (B) at 24 hpi. (C) Schematic diagram showing genomic region of pDCP1a gene and the target site used for CRISPR/Cas9 gRNA design. Sequencing analysis of genomic DNA from the isolated pDCP1a-knockout (KO) cells. (D) Western blot analysis of en- dogenous pDCP1a protein levels in cell lysates from pDCP1a-WT or pDCP1a-KO cells. (E and F) pDCP1a and its mutants were transfected into PK-15CD163-DCP1aKO cells, respectively. At 24 h posttransfection, cells were infected with PRRSV (MOI = 0.5). At 24 hpi, cells were harvested to determine PRRSV RNA by qRT-PCR (E) or PRRSV titers by TCID50 assay (F). All the data were conﬁrmed by three independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001. hpi, hour postinfection; N.S., not signiﬁcant; WT, wild type. by guest on September 25, 2021 demonstrate that PRRSV can directly cleave pDCP1a, revealing a signaling pathway to antagonize IFN production but also cleaves new strategy of PRRSV nsp4–mediated immune evasion. In ISGs to further dampen host defense. It should be noted that no combination with previous and present studies, we can conclude evident cleavage products of IPS-1 and NEMO were observed in that PRRSV nsp4 not only targets key molecules of the type I IFN the context of PRRSV infection in previous studies, although clear

FIGURE 7. PRRSV nsp4 does not cleave mDCP1a. (A) Comparison of aa 238 (indicated by an asterisk) of DCP1a in different species. (B) MARC-145 cells were infected with PRRSV (MOI = 0.5) for the indicated times, and then cell lysates were prepared for Western blot analysis to detect mDCP1a and PRRSV N protein. (C) HEK- 239T cells were transfected with mDCP1a or pDCP1a along with PRRSV nsp4. Cells were lysed and analyzed by Western blotting at 30 h posttransfection. b-Actin was used as a loading control. (D and E) MARC-145 cells were transfected mDCP1a or pDCP1a for 24 h and were subsequently challenged with PRRSV (MOI = 0.5). At 24 hpi, PRRSV RNA was analyzed by qRT-PCR

(D), and virus titers were determined by TCID50 assay (E). Data from three independent experiments are shown (mean 6 SD). *p , 0.05, **p , 0.01. hpi, hour postinfection. 8 PRRSV nsp4 CLEAVES PORCINE DCP1a cleavage bands of IPS-1 and NEMO were detected in cells over- (PRRSV): pathogenesis and interaction with the immune system. Annu. Rev. Anim. Biosci. 4: 129–154. expressing PRRSV nsp4 (54, 56). On the contrary, pDCP1a cleavage 3. Cavanagh, D. 1997. Nidovirales: a new order comprising coronaviridae and products could be clearly detected in both nsp4-overexpressing cells arteriviridae. Arch. Virol. 142: 629–633. and PRRSV-infected cells. In addition, Dougherty et al. (57) found 4. Snijder, E. J., M. Kikkert, and Y. Fang. 2013. Arterivirus molecular biology and pro pathogenesis. J. Gen. Virol. 94: 2141–2163. that both poliovirus 3C cleavage and proteasomal turnover led to 5. Kappes, M. A., and K. S. Faaberg. 2015. PRRSV structure, replication and recom- loss of hDCP1a during poliovirus infection. However, in our present bination: origin of phenotype and genotype diversity. Virology 479–480: 475–486. study, PRRSV nsp4 cleaves pDCP1a independent of the ubiquitin 6. Li, Y., A. Tas, E. J. Snijder, and Y. Fang. 2012. Identification of porcine reproductive and respiratory syndrome virus ORF1a-encoded non-structural pro- proteasome, apoptotic, and autophagy–lysosome pathways, which teins in virus-infected cells. J. Gen. Virol. 93: 829–839. differs from poliovirus 3Cpro–mediated hDCP1a cleavage. 7. Yuan, S., M. P. Murtaugh, F. A. Schumann, D. Mickelson, and K. S. Faaberg. Previous studies showed that DCP1a can induce phosphorylation of 2004. Characterization of heteroclite subgenomic RNAs associated with PRRSV infection. Virus Res. 105: 75–87. eIF2a by activating PKR. Phosphorylated eIF2a leads to translational 8. Fang, Y., and E. J. Snijder. 2010. The PRRSV replicase: exploring the multi- arrest to inhibit poliovirus replication (36). We found that over- functionality of an intriguing set of nonstructural proteins. Virus Res. 154: 61–76. 9. Li, Y., A. Tas, Z. Sun, E. J. Snijder, and Y. Fang. 2015. Proteolytic processing of expression of pDCP1a also triggered PKR activation and increased the porcine reproductive and respiratory syndrome virus replicase. Virus Res. the phosphorylation levels of eIF2a (data not shown), suggesting that 202: 48–59. pDCP1a-induced translational arrest participates in inhibiting PRRSV 10. Kimman, T. G., L. A. Cornelissen, R. J. Moormann, J. M. Rebel, and N. Stockhofe-Zurwieden. 2009. Challenges for porcine reproductive and respi- replication. DCP1a mainly comprises several regions, including an ratory syndrome virus (PRRSV) vaccinology. Vaccine 27: 3704–3718. N-terminal EVH1 domain, a proline-rich region, and a C-terminal 11. Murtaugh, M. P., and M. Genzow. 2011. Immunological solutions for treatment trimerization domain (TD) (58, 59). The TD deletion mutant induces and prevention of porcine reproductive and respiratory syndrome (PRRS). Vaccine 29: 8192–8204. a high level of eIF2a phosphorylation similar to the full-length DCP1a, 12. Meng, X. J. 2000. Heterogeneity of porcine reproductive and respiratory syn- Downloaded from whereas neither DCP1a lacking the EVH1 domain nor the EVH1 drome virus: implications for current vaccine efficacy and future vaccine de- domain alone can induce eIF2a phosphorylation. These observations velopment. Vet. Microbiol. 74: 309–329. 13. Vu, H. L. X., A. K. Pattnaik, and F. A. Osorio. 2017. Strategies to broaden the suggest that the EVH1 domain and proline-rich region are related to cross-protective efficacy of vaccines against porcine reproductive and respiratory DCP1a-induced eIF2a phosphorylation (36). Interestingly, we found syndrome virus. Vet. 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and respiratory syndrome virus. Viruses 7: 873–886. protects piglets against HP-PRRS. Vet. Microbiol. 138: 34–40. by guest on September 25, 2021