Nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15

Abstract

Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.

Fig 1

ISG15 overexpression inhibits PRRSV replication. MARC-145 cells were transfected with different doses of control vector or p3xFLAG-ISG15 plasmid DNA for 48 h. Cells were subsequently infected with PRRSV strain SD01-08 at an MOI of 1 for 24 h. (A) Western blot analysis of ISG15 expression in transfected cells. ISG15 was detected by anti-Flag antibody, and β-tubulin (loading control) was detected using a β-tubulin-specific MAb. (B) Quantitative analysis of ISG15 expression levels. Fold increase in ISG protein levels was determined by comparison with the protein expression level from cells transfected with 0.2 μg of plasmid DNA. (C) Cells were stained with PRRSV N protein specific MAb SDOW17, and FITC-conjugated goat anti-mouse IgG was used as a secondary antibody. Pictures were taken with an Olympus IX71 fluorescence microscope.

Fig 2

Effect of ISG15 conjugation on PRRSV replication. BHK-21 cells were transfected with plasmids that express conjugation enzymes E1/E2/E3 and Flag-tagged ISG15 or ISG15 mutant (ISG15AA). Empty vector plasmid p3xFlag was used as a negative control. At 6 h posttransfection, cells were transfected with the infectious PRRSV cDNA clone pCMV-SD01-08. Cells and supernatant were harvested at 24 h posttransfection. (A) Cell lysates were analyzed by immunoblotting using antibodies that recognize the Flag-tag of ISG15, β-tubulin, or PRRSV nsp1β. (B) Virus titers were determined by fluorescent-focus assay using cell culture supernatant. The results are mean values from four replicates, and virus titers were expressed as numbers of fluorescent-focus units per milliliter (FFU/ml).

Fig 3

PRRSV suppresses ISG15 expression and conjugation. (A) MARC-145 cells were infected with MOI 0.1 of PRRSV SD01-08 or were mock infected. Cells were harvested at 12, 24, 36, or 48 hpi and analyzed by Western blotting. Membranes were probed with a specific MAb to nsp1β or ISG15. (B) Quantification of ISG15 and nsp1β protein levels at different time points postinfection. Fold change in protein levels was determined by comparing with the protein expression level at 12 hpi. (C) Cell culture supernatant was harvested at 12, 24, 36, or 48 hpi. Virus titers were determined by the fluorescent-focus assay on MARC-145 cells. (D) PAMs were infected with PRRSV SD01-08 at an MOI of 0.1 or mock infected. At 24 h postinfection, cells were stimulated with 1,000 U/ml swine IFN-α. Cells were harvested at 24 h poststimulation and analyzed by Western blotting. Membranes were probed with a specific MAb to nsp1β or tubulin (loading control) or a rabbit polyclonal antiserum to swine ISG15.

Fig 4

The PRRSV PLP2-DUB domain inhibits ISG15 expression and conjugation. (A) HeLa cells were transfected with an expression plasmid for nsp2(386–578) or an empty plasmid vector and stimulated with 300 HA/ml of SeV at 24 h posttransfection. Cells were harvested at 24 h poststimulation, and lysates were immunoblotted for detection of ISG15 or nsp2(386–578). An anti-ISG15 MAb was used to detect ISG15 expression, and an anti-nsp2 MAb was used to detect the expression of nsp2(386–578). (B) HeLa cells were cotransfected with plasmid DNAs expressing conjugation enzymes E1/E2/E3, ISG15, and nsp2(386-578). The empty pCAGGS vector plasmid was included as a control. At 6 h posttransfection, cells were stimulated with 1,000 U/ml of IFN-β. Cells were harvested at 24 h poststimulation and analyzed by immunoblotting. The membrane was probed with anti-PRRSV nsp2 MAb to detect the expression of nsp2(386-578). Free and conjugated forms of ISG15 were detected by anti-ISG15 polyclonal antiserum. The anti-β-tubulin antibody was used to detect the expression of β-tubulin (loading control).

Fig 5

Schematic diagram of PRRSV nsp2 N-terminal domain mutations and deletions. Each mutant was constructed in both the pCAGGS expression vector and in PRRSV full-length cDNA clone pCMV-SD01-08. The CD23, CD19, and CD19+1S mutations were also transferred to the pLnsp2-3 plasmid backbone. The arrowheads represent the sites being cleaved by the proteases between nsp1β/2 and nsp2/3. +, viable; −, nonviable. Amino acid numbers refer to the pp1a polyprotein sequence of PRRSV strain SD01-08 (GenBank accession number DQ489311) (13).

Fig 6

Effect of mutations and/or deletions on the ability of PLP2-DUB domain to inhibit IFN-β induction. HeLa cells were transfected with a plasmid that expresses a wild-type or mutated nsp2(386–578), pCAGGS empty vector plasmid, or a plasmid expressing influenza virus NS1 (positive control), along with the reporter plasmid p125-Luc and Renilla luciferase expression plasmid pRL-SV40. Cells were stimulated with Sendai virus at 24 h posttransfection. The luciferase activity was measured at 16 h poststimulation. Relative luciferase activity is defined as the ratio of firefly luciferase reporter activity to Renilla luciferase activity. Each data point represents a mean value from three experiments. Error bars show standard deviations of the normalized data.

Fig 7

Growth characterization of recombinant PRRSVs carrying a 19-aa deletion with and without an additional S462A substitution in the nsp2 N-terminal domain. (A) Plaque morphology of recombinant viruses and wild-type virus. Confluent cell monolayers were infected with 10-fold serial dilutions of wild-type or recombinant virus. At 2 h postinfection, medium was removed and an agar overlay was applied. After 4 days incubation at 37°C, cells were stained using 0.1% crystal violet. (B) Growth kinetics of recombinant viruses in comparison with wild-type virus. MARC-145 cells were infected with each virus at an MOI of 0.5, and the amount of virus produced at 12, 24, 36, 48, 60, and 72 h postinfection was determined by FFA in MARC-145 cells. The results shown are mean values from three replicates.

Fig 8

Effect of PRRSV nsp2 N-terminal domain mutations on ISG15 expression and conjugation. (A) HeLa cells were transfected with an expression plasmid for wild-type and mutant nsp2(386–578), as indicated at the top of the panel. Cells were stimulated with 300HA/ml of SeV at 24 h posttransfection and harvested at 24 h poststimulation. Expression of ISG15 and nsp2(386–578) was analyzed by Western blot. Anti-ISG15 MAb was used to detect the expression of ISG15, and anti-nsp2 polyclonal antibody was used to detect the expression of nsp2(386–578). (B) Quantitative analysis of ISG15 protein expression. Fold increase in ISG15 levels was determined by comparing with the protein expression level from cells transfected with wild-type pCAGGS-nsp2(386–578). (C) Plasmids expressing conjugation enzymes E1/E2/E3 and ISG15 were cotransfected with a plasmid expressing the wild type nsp2(386–578), nsp2 mutant, or a pCAGGS vector control in HeLa cells. Cells were stimulated with 1,000 U/ml IFN-β at 6 h posttransfection. At 24 h poststimulation, cells were harvested for Western blot analysis. Free or conjugated form of ISG15 was detected by rabbit anti-Flag polyclonal antibody.

Fig 9

Effect of nsp2 N-terminal domain mutations on the proteolytic activity toward the nsp2/3 site. RK-13 cells were transfected with plasmids expressing wild-type or mutant nsp2-3 polyproteins, as indicated at the top of the panel. After metabolic labeling, expression products were immunoprecipitated with a rabbit antiserum recognizing both nsp2 and nsp3. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The positions of the nsp2-3 precursor and its cleavage products are indicated on the right.