Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9’s Antiviral Effect via Nsp11 Endoribonuclease Activity

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry worldwide. Our previous study had indicated that proprotein convertase subtilisin/kexin type 9 (PCSK9) was a responsive gene in porcine alveolar macrophages (PAMs) upon PRRSV infection. However, whether PCSK9 impacts the PRRSV replication and how the PRRSV modulates host PCSK9 remains elusive. Here, we demonstrated that PCSK9 protein suppressed the replication of both type-1 and type-2 PRRSV species. More specifically, the C-terminal domain of PCSK9 was responsible for the antiviral activity. Besides, we showed that PCSK9 inhibited PRRSV replication by targeting the virus receptor CD163 for degradation through the lysosome. In turn, PRRSV could down-regulate the expression of PCSK9 in both PAMs and MARC-145 cells. By screening the nonstructural proteins (nsps) of PRRSV, we showed that nsp11 could antagonize PCSK9’s antiviral activity. Furthermore, mutagenic analyses of PRRSV nsp11 revealed that the endoribonuclease activity of nsp11 was critical for antagonizing the antiviral effect of PCSK9. Collectively, our data provide further insights into the interaction between PRRSV and the cell host and offer a new potential target for the antiviral therapy of PRRSV.


Reagents and Antibodies
The monoclonal antibody against the PRRSV N protein was a kind present from Dr. Ying Fang (Department of Animal Sciences and Industry, Kansas State University, Manhattan). Monoclonal antibodies against PRRSV nsp2, PCSK9, and CD163 were produced and stored by our lab. Goat anti-mouse IgG (H + L) antibody conjugated with Alexa Fluor 488 (1:800) was purchased from Abcam (Abcam, Shanghai, China). MG132 and CQ were purchased from Sigma (Sigma, Shanghai, China). DMSO was purchased from MP Biomedicals (MP Biomedicals, Shanghai, China). Lipofectamine ® 3000 Transfection Kits were purchased from Invitrogen (Invitrogen, Shanghai, China). Dual Luciferase Reporter Assay Kits were purchased from Vazyme Biotechnology (Vazyme Biotechnology, Nanjing, China). Monoclonal Anti-HA-Agarose antibody produced in mice was purchased from Sigma (Sigma, Shanghai, China). PrimeSTAR ® HS DNA Polymerase with GC Buffer was purchased from Takara (Takara, Dalian, China). DAPI was purchased from Beyotime (Beyotime, Shanghai, China).

Multi-Step Growth Curve of Virus
MARC-145 cells cultured in 6-well plates were transfected with either pCAGGS-PCSK9-Flag or the empty vector pCAGGS in triplicate and then infected with PRRSV at an MOI (Multiplicity of infection) of 0.1 at 36 h after transfection. Then, 250 µL of supernatant was collected at different time points post infection, followed by the addition of 250 µL of fresh DMEM with 2% FBS (Gibco, Australia). One hundred microliters of supernatant was used for the TCID50 assay.

TCID50 Assay for PRRSV
MARC-145 cells were seeded in 96-well plates and then infected with serial 10-fold dilutions of PRRSV samples in eight replicates. Ninety-six hours later, the virus titers were calculated based on the Reed-Muench method.

RNA Extraction and RT-qPCR Assay
For virus copy number detection, total RNA was extracted from cultured cells with the RNeasy ® Mini Kit (QIAGEN, Hilden, Germany), and the RNA in the supernatant was extracted with the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). The RNA was then reverse transcribed into cDNA with a reverse transcriptase mix (Takara, Dalian, China). For PAM RNA extraction, cells were washed using PBS three times before RNA extraction. To each sample was added 333 uL of TRIzol (Thermo Fisher Scientific, Shanghai, China). Three samples were merged into one sample, and the total RNA was extracted and then reverse transcribed into cDNA with reverse transcriptase (Thermo Fisher Scientific, Shanghai, China). Quantitative real-time PCR experiments were performed in triplicate. The relative level of mRNA expression was normalized to that of GAPDH. Absolute quantitative mRNA levels were calculated using standard curves.

Co-Immunoprecipitation (Co-IP)
Co-IP was performed as described previously [33]. Briefly, HEK-293T cells were co-transfected with the indicated plasmid using Lipofectamine 3000 Transfection Kits (Invitrogen, Shanghai, China). Twenty-four hours after transfection, the lysates were collected with the IP lysis buffer (Thermo Fisher Scientific, Shanghai, China), and then 15 µL of Agarose beads with monoclonal antibody against HA or Flag (Sigma, Shanghai, China) were added to each sample and incubated at 4 • C for 6 h with rotation. Then, the beads were pelleted and washed 5 times with IP lysis buffer. Finally, the proteins were dissolved in 50 µL of IP lysis buffer.

Reporter Assay
HEK-293T cells were seeded in 12-well plates and transfected with related plasmids when the cells reached 70% confluence, and they were transfected with a mixture of IFN-β-luc and pRL-TK-Renilla luciferase plasmids and appropriate control or protein-expressing plasmid(s). Twenty-four hours after transfection, the cells were treated with regent poly (I:C) (Invivogen, Shanghai, China) for the IFN stimulation and then collected at 12 h after treatment. Reporter gene activity was determined by the normalization of firefly luciferase activity against Renilla luciferase activity.

Bioinformatics Prediction
The I-TASSER online service tool was used to predict the 3D structure of porcine PCSK9, referring to the human PCSK9 protein (Protein Data Bank, ID: 2P4E). The 3D structure of porcine PCSK9 was visualized using the VMD software (Version 1.9.4, IL, USA).

Statistical Analyses
All data were analyzed with GraphPad Prism 5 (GraphPad, San Diego, CA, USA) and are provided as the mean ± SEM unless otherwise specified in figure legends. Statistical analyses were performed using the unpaired two-tailed Student's t-test. Differences between groups were considered statistically significant when the p value was less than 0.05 (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). The gray values from Western blotting were calculated using the Image J software (https://imagej.nih.gov/ij/).

PCSK9 Inhibits the Replication of Both Type-1 and Type-2 PRRSV Species
In our previous study, we performed a liquid chromatography-tandem mass spectrometry (LS-MS/MS) analysis of PAMs infected with or without the PRRSV strain HuN4-eGFP. The LS-MS/MS result showed that the PCSK9 expression level increased significantly upon PRRSV strain HuN4-eGFP infection in PAMs compared to in the mock infected PAMs [47]. However, the function of PCSK9 in PRRSV replication is not yet defined. To this end, we overexpressed PCSK9 in MARC-145 cells by transfecting a vector expressing porcine PCSK9 to evaluate the effect of PCSK9 on PRRSV replication. Firstly, the PCSK9-transfected MARC-145 cells were infected with the highly pathogenic PRRSV strain HuN4, and then, the expression of the PRRSV nsp2 protein was analyzed by immunofluorescence. As shown in Figure 1A, the fluorescence signal was significantly lower in pCAGGS-PCSK9-Flag-transfected cells compared to that in the empty vector pCAGGS-transfected cells. In addition, we collected virus-containing supernatant after HuN4 strain infection from pCAGGS-PCSK9-Flag-and pCAGGS-transfected MARC-145 cells at different time points. Compared to those in control, the virus titers from the PSCK9-overexpressing cells were much lower ( Figure 1B). We further confirmed this finding by investigating the expression level of the PCSK9 protein and PRRSV N protein using Western blotting (WB) ( Figure 1C). Taken together, these data indicated that PCSK9 inhibited PRRSV replication in vitro. Then, we reasoned that PCSK9 could suppress not only type-2 PRRSV replication but also type-1 PRRSV replication. Therefore, we infected PCSK9-overexpressing cells with the type-1 PRRSV strain Lelystad as well as the type-2 PRRSV strains APRRSV, HuN4, and F112 and collected the supernatants from the infected cells. The virus titers decreased in PCSK9-overexpressing cells compared to those in the control cells for both types of PRRSV ( Figure 1d). This result demonstrated that the ectopic expression of PCSK9 led to the suppression of both type-1 and type-2 PRRSV replication.

The C-Terminal Domain of PCSK9 Has Antiviral Activity
The human PCSK9 protein consists of a signal sequence followed by a pro-domain, a catalytic domain, and a C-terminal domain. The PCSK9 protein is synthesized as an inactive pro-enzyme and contains a triad of residues (Asp-186, His-226, and Ser-386) that are required for catalytic activity [42,48]. Human PCSK9 has been well studied since it is a key player in plasma cholesterol metabolism. However, there is limited information available about the porcine PCSK9 protein. Thus, we compared the human and porcine PCSK9 protein structures and found that porcine PCSK9 contained similar domains to human PCSK9 (Figure 2a). We also obtained the residues that were possibly responsible for porcine PCSK9 protein maturation (Figure 2a). To confirm if those residues were involved in PCSK9 pre-protein catalysis, we generated a set of single amino acid-mutated Then, we reasoned that PCSK9 could suppress not only type-2 PRRSV replication but also type-1 PRRSV replication. Therefore, we infected PCSK9-overexpressing cells with the type-1 PRRSV strain Lelystad as well as the type-2 PRRSV strains APRRSV, HuN4, and F112 and collected the supernatants from the infected cells. The virus titers decreased in PCSK9-overexpressing cells compared to those in the control cells for both types of PRRSV ( Figure 1D). This result demonstrated that the ectopic expression of PCSK9 led to the suppression of both type-1 and type-2 PRRSV replication.

The C-Terminal Domain of PCSK9 Has Antiviral Activity
The human PCSK9 protein consists of a signal sequence followed by a pro-domain, a catalytic domain, and a C-terminal domain. The PCSK9 protein is synthesized as an inactive pro-enzyme and contains a triad of residues (Asp-186, His-226, and Ser-386) that are required for catalytic activity [42,48]. Human PCSK9 has been well studied since it is a key player in plasma cholesterol metabolism. However, there is limited information available about the porcine PCSK9 protein. Thus, we compared the human and porcine PCSK9 protein structures and found that porcine PCSK9 contained similar domains to human PCSK9 (Figure 2A). We also obtained the residues that were possibly responsible for porcine PCSK9 protein maturation (Figure 2A). To confirm if those residues were involved in PCSK9 pre-protein catalysis, we generated a set of single amino acid-mutated variants of the PCSK9 protein ( Figure 2A). The WB result showed that residues 197, 237, 328, and 397 were required for PCSK9 protein maturation, whereas residue 150 was not necessary ( Figure 2B). variants of the PCSK9 protein (Figure 2a). The WB result showed that residues 197, 237, 328, and 397 were required for PCSK9 protein maturation, whereas residue 150 was not necessary (Figure 2b). We speculated that mature PCSK9, but not immature PCSK9, possessed antiviral activity. Therefore, we examined the antiviral activity of these mutated PCSK9 proteins by overexpressing these mutants in MARC-145 cells, followed by PRRSV strain HuN4 infection. Interestingly, the WB analysis of the PCSK9 protein and PRRSV N protein showed that a single residue mutation in PCSK9 could not affect the antiviral activity of PCSK9 ( Figure 2c). Furthermore, to determine which domain of PCSK9 protein is crucial for the antiviral activity, we constructed several PCSK9 variants with peptides truncated then overexpressed these truncated PCSK9 proteins in MARC-145 cells, followed by PRRSV strain HuN4 infection. The WB result showed that the C-terminal domain of PCSK9 was sufficient to inhibit PRRSV replication (Figure 2d).

PCSK9 Degrades PRRSV Receptor CD163 Through Lysosome Pathway
The function of PCSK9 is the binding of specific cell surface receptors to bring them towards the intracellular lysosome degradation compartments [49,50]. In addition to reducing the receptors through the lysosome, PCSK9 also can directly interact with CD36 and target the receptor to lysosomes through a mechanism involving the proteasome [51]. These findings suggest that PCSK9 may inhibit PRRSV replication through regulating the virus receptor presenting on target cells through the lysosomal and/or proteasomal pathway. We tested the mRNA abundance of several receptors-CD163, CD151, and vimentin-upon PCSK9 overexpression in MARC-145 cells. As expected, none of these receptors showed a significant difference in terms of mRNA amounts after PCSK9 overexpression (Figure 3a), suggesting that PCSK9 could regulate CD163 post translation. Therefore, HEK-293T cells were transfected with pCAGGS-CD163-HA and pCAGGS-PCSK9-Flag or pCAGGS vector. The cell lysates were analyzed by WB. Compared to control, the level of CD163 We speculated that mature PCSK9, but not immature PCSK9, possessed antiviral activity. Therefore, we examined the antiviral activity of these mutated PCSK9 proteins by overexpressing these mutants in MARC-145 cells, followed by PRRSV strain HuN4 infection. Interestingly, the WB analysis of the PCSK9 protein and PRRSV N protein showed that a single residue mutation in PCSK9 could not affect the antiviral activity of PCSK9 ( Figure 2C). Furthermore, to determine which domain of PCSK9 protein is crucial for the antiviral activity, we constructed several PCSK9 variants with peptides truncated then overexpressed these truncated PCSK9 proteins in MARC-145 cells, followed by PRRSV strain HuN4 infection. The WB result showed that the C-terminal domain of PCSK9 was sufficient to inhibit PRRSV replication ( Figure 2D).

PCSK9 Degrades PRRSV Receptor CD163 Through Lysosome Pathway
The function of PCSK9 is the binding of specific cell surface receptors to bring them towards the intracellular lysosome degradation compartments [49,50]. In addition to reducing the receptors through the lysosome, PCSK9 also can directly interact with CD36 and target the receptor to lysosomes through a mechanism involving the proteasome [51]. These findings suggest that PCSK9 may inhibit PRRSV replication through regulating the virus receptor presenting on target cells through the lysosomal and/or proteasomal pathway. We tested the mRNA abundance of several receptors-CD163, CD151, and vimentin-upon PCSK9 overexpression in MARC-145 cells. As expected, none of these receptors showed a significant difference in terms of mRNA amounts after PCSK9 overexpression ( Figure 3A), suggesting that PCSK9 could regulate CD163 post translation. Therefore, HEK-293T cells were transfected with pCAGGS-CD163-HA and pCAGGS-PCSK9-Flag or pCAGGS vector. The cell lysates were analyzed by WB. Compared to control, the level of CD163 significantly decreased in PCSK9-overexpressing cells ( Figure 3B). To further examine whether the PCSK9 protein could bind to CD163, HEK-293T cells were co-transfected with pCAGGS-CD163-HA and/or pCAGGS-PCSK9-Flag.
The cells were lysed for immunoprecipitation with an antibody against HA tag, followed by WB analysis with antibodies against Flag and HA tag. As shown in Figure 3C, the level of PCSK9 protein in the presence of CD163 was significantly higher than that of the empty vector, whereas there were similar levels of the PCSK9 protein in the input with or without CD163 protein. We further confirmed the interaction between PCSK9 and CD163 by immunoprecipitating with an antibody against Flag tag followed by WB analysis with antibodies against Flag and HA tag ( Figure 3D). Furthermore, colocalization studies were conducted by co-transfecting HeLa cells with pCAGGS-CD163-HA and/or pCAGGS-PCSK9-Flag. Immunofluorescence analysis showed that PCSK9 and CD163 colocalized both in the cytoplasm and on the cell membrane ( Figure 3E). significantly decreased in PCSK9-overexpressing cells (Figure 3b). To further examine whether the PCSK9 protein could bind to CD163, HEK-293T cells were co-transfected with pCAGGS-CD163-HA and/or pCAGGS-PCSK9-Flag. The cells were lysed for immunoprecipitation with an antibody against HA tag, followed by WB analysis with antibodies against Flag and HA tag. As shown in Figure 3c, the level of PCSK9 protein in the presence of CD163 was significantly higher than that of the empty vector, whereas there were similar levels of the PCSK9 protein in the input with or without CD163 protein. We further confirmed the interaction between PCSK9 and CD163 by immunoprecipitating with an antibody against Flag tag followed by WB analysis with antibodies against Flag and HA tag (Figure 3d). Furthermore, colocalization studies were conducted by co-transfecting HeLa cells with pCAGGS-CD163-HA and/or pCAGGS-PCSK9-Flag. Immunofluorescence analysis showed that PCSK9 and CD163 colocalized both in the cytoplasm and on the cell membrane (Figure 3e).  We speculated that PCSK9 could degrade CD163 protein through the proteasome or lysosome. For this purpose, we cotransfected HEK-293T cells with pCAGGS-CD163-HA and pCAGGS-PCSK9-Flag then treated the cells with DMSO or the proteasomal inhibitor MG132 or lysosomal inhibitor chloroquine (CQ). The cell lysates were analyzed by WB with antibodies against HA and Flag tag. For the proteasomal pathway, compared to the DMSO control, CD163 abundance slightly increased in the MG132-treated cells with different doses ( Figure 3F). For the lysosomal pathway, the WB result showed that lysosome inhibition resulted in a higher level of the CD163 protein compared to that following DMSO treatment ( Figure 3F). This finding indicates that the lysosome pathway involves the degradation of CD163 by the PCSK9 protein.

PCSK9 Promotes Interferon Production in a Dose-Dependent Manner
It has been reported that PCSK9 decreases IFN-β promoter/enhancer activity to suppress IFN-β production in humans [52]. To confirm whether the porcine PCSK9 could regulate the innate immunity response, we tested the effect of porcine PCSK9 on IFN-β production in MARC-145 cells. Unlike the effect of PCSK9 on IFN-β production in human cells, the qPCR result showed that PCSK9 promoted the production of IFN-β ( Figure 4A). These contradictory results might be due to the different settings in the cell lines. To further investigate if PCSK9 could bind to the promoter region of the IFN-β gene and regulate the transcription of PCSK9, we measured IFN-β gene promoter activity in PCSK9-overexpressing HEK-293T cells. The result showed that PCSK9 enhanced the activity of the promoter of the IFN-β gene to regulate IFN-β transcription ( Figure 4B). We speculated that PCSK9 could degrade CD163 protein through the proteasome or lysosome. For this purpose, we cotransfected HEK-293T cells with pCAGGS-CD163-HA and pCAGGS-PCSK9-Flag then treated the cells with DMSO or the proteasomal inhibitor MG132 or lysosomal inhibitor chloroquine (CQ). The cell lysates were analyzed by WB with antibodies against HA and Flag tag. For the proteasomal pathway, compared to the DMSO control, CD163 abundance slightly increased in the MG132-treated cells with different doses (Figure 3f). For the lysosomal pathway, the WB result showed that lysosome inhibition resulted in a higher level of the CD163 protein compared to that following DMSO treatment (Figure 3f). This finding indicates that the lysosome pathway involves the degradation of CD163 by the PCSK9 protein.

PCSK9 Promotes Interferon Production in a Dose-Dependent Manner
It has been reported that PCSK9 decreases IFN-β promoter/enhancer activity to suppress IFN-β production in humans [52]. To confirm whether the porcine PCSK9 could regulate the innate immunity response, we tested the effect of porcine PCSK9 on IFN-β production in MARC-145 cells. Unlike the effect of PCSK9 on IFN-β production in human cells, the qPCR result showed that PCSK9 promoted the production of IFN-β (Figure 4a). These contradictory results might be due to the different settings in the cell lines. To further investigate if PCSK9 could bind to the promoter region of the IFN-β gene and regulate the transcription of PCSK9, we measured IFN-β gene promoter activity in PCSK9-overexpressing HEK-293T cells. The result showed that PCSK9 enhanced the activity of the promoter of the IFN-β gene to regulate IFN-β transcription (Figure 4b).

PRRSV Down-Regulates PCSK9 Expression Both in MARC-145 Cells and in PAM Cells
To examine the possible influences of PRRSV on PCSK9 expression, we infected MARC-145 cells with different doses of the PRRSV strain HuN4. The RT-qPCR results showed that endogenous PCSK9 mRNA expression increased significantly compared to that in the mock control when the cells were infected with a low dose of the virus (Figure 5a). Interestingly, the PCSK9 expression level is comparable to that in the mock control when they are infected with a high dose of the virus (Figure 5a). To further examine the PCSK9 expression changes during PRRSV infection, we infected MARC-145 cells with HuN4 and quantified PCSK9 expression using RT-qPCR. The PCSK9 expression level decreased as the time increased after infection (Figure 5b). We confirmed this finding with PAM cells; as shown in Figure 5C, PRRSV reduces the PCSK9 expression level in a

PRRSV Down-Regulates PCSK9 Expression Both in MARC-145 Cells and in PAM Cells
To examine the possible influences of PRRSV on PCSK9 expression, we infected MARC-145 cells with different doses of the PRRSV strain HuN4. The RT-qPCR results showed that endogenous PCSK9 mRNA expression increased significantly compared to that in the mock control when the cells were infected with a low dose of the virus ( Figure 5A). Interestingly, the PCSK9 expression level is comparable to that in the mock control when they are infected with a high dose of the virus ( Figure 5A). To further examine the PCSK9 expression changes during PRRSV infection, we infected MARC-145 cells with HuN4 and quantified PCSK9 expression using RT-qPCR. The PCSK9 expression level decreased as the time increased after infection ( Figure 5B). We confirmed this finding with PAM cells; as shown in Figure 5C, PRRSV reduces the PCSK9 expression level in a time-dependent manner. We further examined the PCSK9 protein level at various time points in MARC-145 cells that were transfected with PCSK9 followed by HuN4 virus infection. The WB result showed that the PCSK9 protein level decreased as the infection time increased compared to that in the mock infection control ( Figure 5D). A similar result was observed in PAMs; endogenous PCSK9 expression decreased as the infection time increased, whereas that of the PRRSV N protein did the opposite ( Figure 5E).
Viruses 2020, 12, x FOR PEER REVIEW  12 of 19 time-dependent manner. We further examined the PCSK9 protein level at various time points in MARC-145 cells that were transfected with PCSK9 followed by HuN4 virus infection. The WB result showed that the PCSK9 protein level decreased as the infection time increased compared to that in the mock infection control (Figure 5d). A similar result was observed in PAMs; endogenous PCSK9 expression decreased as the infection time increased, whereas that of the PRRSV N protein did the opposite (Figure 5e).

PRRSV nsp11 Negatively Regulates PCSK9 Expression
PCSK9 expression increased in the early stage of PRRSV infection, but the increase was suppressed in the late stages of infection, suggesting that proteins produced during PRRSV replication could negatively regulate PCSK9 expression. To explore which nsps of PRRSV were

PRRSV nsp11 Negatively Regulates PCSK9 Expression
PCSK9 expression increased in the early stage of PRRSV infection, but the increase was suppressed in the late stages of infection, suggesting that proteins produced during PRRSV replication could Viruses 2020, 12, 655 13 of 18 negatively regulate PCSK9 expression. To explore which nsps of PRRSV were involved in the down-regulation of PCSK9 expression, several nsps including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 of PRRSV were cloned and cotransfected with pCAGGS-PCSK9-Flag into HEK-293T cells. At 24 hpt, cell lysates were collected and analyzed by WB. The PCSK9 expression level was significantly decreased by PRRSV nsp11, whereas the effects of other PRRSV nsps on PCSK9 expression were not obvious ( Figure 6A,B). Moreover, we cotransfected HEK-293T cells with different amounts of pCAGGS-NSP11-HA and the same amount of the pCAGGS-PCSK9-Flag plasmid. We found that nsp11 down-regulated PCSK9 expression in a dose-dependent manner ( Figure 6C). These data suggest that nsp11 could antagonize the antiviral function of PCSK9 during PRRSV replication.
Viruses 2020, 12, x FOR PEER REVIEW 13 of 19 involved in the down-regulation of PCSK9 expression, several nsps including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 of PRRSV were cloned and cotransfected with pCAGGS-PCSK9-Flag into HEK-293T cells. At 24 hpt, cell lysates were collected and analyzed by WB. The PCSK9 expression level was significantly decreased by PRRSV nsp11, whereas the effects of other PRRSV nsps on PCSK9 expression were not obvious (Figure 6a,b). Moreover, we cotransfected HEK-293T cells with different amounts of pCAGGS-NSP11-HA and the same amount of the pCAGGS-PCSK9-Flag plasmid. We found that nsp11 down-regulated PCSK9 expression in a dose-dependent manner (Figure 6c). These data suggest that nsp11 could antagonize the antiviral function of PCSK9 during PRRSV replication.

PRRSV nsp11 Inhibits PCSK9 via Its Endoribonuclease Activity
In light of the significant consequences of the nsp11-mediated negative regulation of PCSK9 expression, we further explored the molecular mechanism. Firstly, to examine whether there was an interaction between the nsp11 and PCSK9 proteins, cells were co-expressed with pCAGGS-HA-Nsp11 and pCAGGS-PCSK9-Flag. Co-IP was performed with an antibody against HA, followed by WB analysis. As shown in Figure 7a, nsp11 did not interact with the PCSK9 protein.
Secondly, to figure out the binding of nsp11 to the promoter regions of PCSK9 to inhibit PCSK9 transcription, we measured PCSK9 promoter activity in nsp11-over-expressing cells. For this purpose, HEK-239T cells were cotransfected with a luciferase reporter containing the PCSK9 promoter with or without pCAGGS-HA-Nsp11. The reporter luciferase activity showed no significant difference between the nsp11-over-expressing cells and the control group (Figure 7b,c), indicating that nsp11 could not regulate PCSK9 transcription by binding to the PCSK9 promoter. Then, we investigated if nsp11 targeted PCSK9 through the proteasome pathway or lysosome pathway. Through the inhibition of the proteasome or lysosome, we confirmed that neither the proteasome nor lysosome was responsible for the down-regulation of PCSK9 (Figure 7d). Finally, we constructed a set of nsp11 constructs containing mutations that could inactivate either the endoribonuclease activity or deubiquitinating activity of nsp11 and cotransfected pCAGGS-PCSK9-Flag with nsp11 mutants into cells. The WB results showed that nsp11 mutants with inactivated endoribonuclease activity lost the function of antagonizing PCSK9 antiviral activity, whereas those nsp11 mutants that lost their deubiquitinating activity did not (Figure 7e). Collectively, these results showed that nsp11 negatively regulated PCSK9 expression through its endoribonuclease activity, not deubiquitinating activity.

PRRSV nsp11 Inhibits PCSK9 via Its Endoribonuclease Activity
In light of the significant consequences of the nsp11-mediated negative regulation of PCSK9 expression, we further explored the molecular mechanism. Firstly, to examine whether there was an interaction between the nsp11 and PCSK9 proteins, cells were co-expressed with pCAGGS-HA-Nsp11 and pCAGGS-PCSK9-Flag. Co-IP was performed with an antibody against HA, followed by WB analysis. As shown in Figure 7A, nsp11 did not interact with the PCSK9 protein. Secondly, to figure out the binding of nsp11 to the promoter regions of PCSK9 to inhibit PCSK9 transcription, we measured PCSK9 promoter activity in nsp11-over-expressing cells. For this purpose, HEK-239T cells were cotransfected with a luciferase reporter containing the PCSK9 promoter with or without pCAGGS-HA-Nsp11. The reporter luciferase activity showed no significant difference between the nsp11-over-expressing cells and the control group ( Figure 7B,C), indicating that nsp11 could not regulate PCSK9 transcription by binding to the PCSK9 promoter. Then, we investigated if nsp11 targeted PCSK9 through the proteasome pathway or lysosome pathway. Through the inhibition of the proteasome or lysosome, we confirmed that neither the proteasome nor lysosome was responsible for the down-regulation of PCSK9 ( Figure 7D). Finally, we constructed a set of nsp11 constructs containing mutations that could inactivate either the endoribonuclease activity or deubiquitinating activity of nsp11 and cotransfected pCAGGS-PCSK9-Flag with nsp11 mutants into cells. The WB results showed that nsp11 mutants with inactivated endoribonuclease activity lost the function of antagonizing PCSK9 antiviral activity, whereas those nsp11 mutants that lost their deubiquitinating activity did not ( Figure 7E). Collectively, these results showed that nsp11 negatively regulated PCSK9 expression through its endoribonuclease activity, not deubiquitinating activity. HEK-293T cells were cotransfected with different combinations of vectors as indicated. Cell lysates were harvested at 24 hpt, and immunoprecipitation was performed using an antibody against HA, followed by WB analysis. (B and C) 855 bp PCSK9 promoter sequence was cloned into the pGL3-Basic vector. pGL3-PCSK9 was cotransfected with pCAGGS-HA-Nsp11(1 μg) or pCAGGS empty vector (1 μg) as a negative control into HEK-293T cells. Error bar: mean ± SEM; ***, p ≤ 0.001.; ns: no significant. (D) HEK-293T cells were transfected with pCAGGS-PCSK9-Flag (2 μg) and/or pCAGGS-HA-Nsp11 (2 μg) as indicated. At 18 hpt, the cells were further treated with/without the proteasome inhibitor MG132 or lysosomal inhibitor chloroquine (CQ) or vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB to assess PCSK9 and nsp11 expression. (E) A set of nsp11 constructs containing mutations that could inactivate endoribonuclease activity (nsp11-H129A, nsp11-H144A, nsp11-K173A, nsp11-H129H144A, and nsp11-C112K173A) or deubiquitinating activity (nsp11-C112A) (2 μg) were generated. pCAGGS-PCSK9-Flag (2 μg) was cotransfected with nsp11 mutants and wild type nsp11 into HEK-293T cells. WB was performed to analyze PCSK9 and nsp11 expression.

Discussion
Viruses regulate host cellular components and try to take control of the normal cell networks to facilitate their survival and replication. Conversely, the cell hosts are responsive to viral infection and fight viruses through a variety of anti-viral approaches. Although the interactions between PRRSV and cells have been extensively studied [2], more research on host changes upon PRRSV infection is needed, because the knowledge of these factors is essential for understanding viral infection and can offer potential targets for antiviral therapies as well as a new insight for vaccine design. In this study, we found that a new antiviral factor PCSK9 could significantly inhibit the replication of PRRSV. We also explored the amino acid of porcine PCSK9 key for PCSK9 maturation and the effect of pre-PCSK9 and mature PCSK9 on PRRSV replication. Intriguingly, both the pre-PCSK9 and mature PCSK9 show antiviral activity against PRRSV. In this case, we hypothesized that a specific domain of PCSK9 instead of whole protein could play an important role in the inhibition of PRRSV replication. Further investigation indicated that the C-terminal domain of PCSK9 has antiviral activity. Cell lysates were harvested at 24 hpt, and immunoprecipitation was performed using an antibody against HA, followed by WB analysis. (B and C) 855 bp PCSK9 promoter sequence was cloned into the pGL3-Basic vector. pGL3-PCSK9 was cotransfected with pCAGGS-HA-Nsp11(1 µg) or pCAGGS empty vector (1 µg) as a negative control into HEK-293T cells. Error bar: mean ± SEM; ***, p ≤ 0.001.; ns: no significant. (D) HEK-293T cells were transfected with pCAGGS-PCSK9-Flag (2 µg) and/or pCAGGS-HA-Nsp11 (2 µg) as indicated. At 18 hpt, the cells were further treated with/without the proteasome inhibitor MG132 or lysosomal inhibitor chloroquine (CQ) or vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB to assess PCSK9 and nsp11 expression. (E) A set of nsp11 constructs containing mutations that could inactivate endoribonuclease activity (nsp11-H129A, nsp11-H144A, nsp11-K173A, nsp11-H129H144A, and nsp11-C112K173A) or deubiquitinating activity (nsp11-C112A) (2 µg) were generated. pCAGGS-PCSK9-Flag (2 µg) was cotransfected with nsp11 mutants and wild type nsp11 into HEK-293T cells. WB was performed to analyze PCSK9 and nsp11 expression.

Discussion
Viruses regulate host cellular components and try to take control of the normal cell networks to facilitate their survival and replication. Conversely, the cell hosts are responsive to viral infection and fight viruses through a variety of anti-viral approaches. Although the interactions between PRRSV and cells have been extensively studied [2], more research on host changes upon PRRSV infection is needed, because the knowledge of these factors is essential for understanding viral infection and can offer potential targets for antiviral therapies as well as a new insight for vaccine design. In this study, we found that a new antiviral factor PCSK9 could significantly inhibit the replication of PRRSV. We also explored the amino acid of porcine PCSK9 key for PCSK9 maturation and the effect of pre-PCSK9 and mature PCSK9 on PRRSV replication. Intriguingly, both the pre-PCSK9 and mature PCSK9 show antiviral activity against PRRSV. In this case, we hypothesized that a specific domain of PCSK9 instead of whole protein could play an important role in the inhibition of PRRSV replication. Further investigation indicated that the C-terminal domain of PCSK9 has antiviral activity.
Several host factors have been discovered that can suppress PRRSV replication, most of them, by targeting the innate immune pathways [2,53]. As for the role of PCSK9, studies suggest that PCSK9 plays a role in immunity modulation, such as by influencing IFN and inflammatory factor production. Our results showed that PCSK9 could increase the mRNA level of IFN-β by influencing its promoter. This finding suggests that PCSK9 may inhibit PRRSV replication in part through up-regulation of the IFN-β product. However, this finding is not in line with the PCSK9 function reported in the previous study, in which IFN-β expression was inhibited in human cells [52]. In the previous study, PCSK9 suppressed IFN-β expression through inhibiting activating transcription factor-2 (ATF-2)/c-Jun dimerization and the binding of ATF-2/c-Jun to the IFN-β enhancer via interaction with ATF-2. However, we showed that PCSK9 increased IFN-β expression by influencing its promoter. The mechanism underlying PCSK9's effect on IFN-β expression needs to be further studied.
PCSK9 can impede HCV replication in human live cells by decreasing the expression of LDLR through binding to LDLR and delivering the PCSK9-LDLR complex to lysosomes for degradation [49,50]. In addition to targeting cell receptors through the lysosomal pathway, evidence shows that PCSK9 can partially target protein degradation through the proteasomal pathway. One study shows that PCSK9 reduces the expression of the CD36 receptor by directly interacting with CD36 and targeting the receptor to lysosomes through a mechanism involving the proteasome [51]. Based on these findings, we probe the effect of PCSK9 on CD163, which is required for PRRSV infection. Similar to CD36 or LDLR degradation by PCSK9 via the lysosome, we showed that PCSK9 could bind to CD163 and decrease the CD163 protein level. These observations imply that PCSK9 inhibited PRRSV replication by delivering the PCSK9-CD163 complex to lysosomes for degradation. However, we cannot rule out other mechanisms of PCSK9's effect on PRRSV replication. The possible pathway underlying the regulation by PCSK9 of PRRSV replication needs to be addressed further.
PRRSV nsp11 possesses both endoribonuclease activity and deubiquitinating activity. The endoribonuclease activity of nsp11 is conserved and unique for viruses in the order Nidovirales. The NendoU domain of PRRSV nsp11 elicits the nuclease activity. PRRSV nsp11 is an IFN antagonist, and the endoribonuclease activity is critical for IFN suppression [53]. PRRSV nsp11 also can target MAVS and degrade the MAVS mRNA, leading to the failure of the activation of downstream pathways to suppress IFN production [19]. Unlike previous findings, we show that nsp11 antagonizes PCSK9's antiviral activity by inducing the degradation of the PCSK9 transcript through its endoribonuclease activity. However, the endoribonuclease activity of nsp11 is not specific. We also confirmed this by overexpressing EGFP and nsp11. Consistent with the previous findings [54], nsp11 could slightly decrease the expression of EGFP (data not shown). The mechanism by which nsp11 selectively degrades PCSK9 RNA needs to be further investigated.
In summary, we have investigated the relationship between PCSK9 and PRRSV replication and revealed that PCSK9 has antiviral activity against PRRSV. The C-terminal domain of PCSK9 plays a central role in its antiviral activity. PCSK9 could bind to CD163 and deliver CD163 to the lysosome for degradation. Besides, we reveal that PRRSV can antagonize the antiviral activity of PCSK9 through nsp11 endoribonuclease activity. Our finding broadens our understanding of how PRRSV nsp11 antagonizes host factors to facilitate viral survival and replication, which provide further insights into the interaction between PRRSV and the cell host, and offer a new antiviral target for curbing the spread of PRRSV.