Targeting Swine Leukocyte Antigen Class I Molecules for Proteasomal Degradation by the nsp1α Replicase Protein of the Chinese Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a critical pathogen of swine, and infections by this virus often result in delayed, low-level induction of cytotoxic T lymphocyte (CTL) responses in pigs. Here, we report that a Chinese highly pathogenic PRRSV strain possessed the ability to downregulate swine leukocyte antigen class I (SLA-I) molecules on the cell surface of porcine alveolar macrophages and target them for degradation in a manner that was dependent on the ubiquitin-proteasome system. Moreover, we found that the nsp1α replicase protein contributed to this property of PRRSV. Further mutagenesis analyses revealed that this function of nsp1α required the intact molecule, including the zinc finger domain, but not the cysteine protease activity. More importantly, we found that nsp1α was able to interact with both chains of SLA-I, a requirement that is commonly needed for many viral proteins to target their cellular substrates for proteasomal degradation. Together, our findings provide critical insights into the mechanisms of how PRRSV might evade cellular immunity and also add a new role for nsp1α in PRRSV infection.

Importance: PRRSV infections often result in delayed, low-level induction of CTL responses in pigs. Deregulation of this immunity is thought to prevent the virus from clearance in an efficient and timely manner, contributing to persistent infections in swineherds. Our studies in this report provide critical insight into the mechanism of how PRRSV might evade CTL responses. In addition, our findings add a new role for nsp1α, a critical viral factor involved in antagonizing host innate immunity.

FIG 1

Infection progression of PAMs by PRRSV. (A and B) PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At the indicated time points, the virus-induced CPE was monitored, and pictures were taken under an inverted microscope. To test cell viability, infected cells were gently washed three times with 1× PBS to remove dead cells and then dissociated from the plates with 0.1% EDTA at 37°C. Cell viability was then determined by trypan blue staining. (C and D) Statistical data.

FIG 2

Downregulation of SLA-I cell surface accumulation of PAMs by HP-PRRSV. (A) Effect of PRRSV on SLA-I cell surface accumulation. PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At the indicated time points, the cells were harvested and subjected to FACS analysis with mouse monoclonal antibody against SLA-I (JM1E3). (B) PRRSV- or mock-inoculated PAMs were incubated with saturating amounts of JM1E3 at 4°C for 1 h before being washed three times with ice-cold PBS to remove unbound MAbs. The cells were then incubated at 37°C to promote endocytosis. At different times postinfection, the cells were collected and subjected to FACS analysis with FITC-conjugated goat anti-mouse IgG. (C) PRRSV- or mock-infected PAMs were incubated with saturating amounts of unlabeled MAb JM1E3 to SLA-I at 4°C for 1 h and then at 37°C for various times before being subjected to FACS analysis with FITC-conjugated JM1E3. The trend curves were fitted based on the MFIs, and the data shown are means and standard deviations of results from three independent experiments (*, P < 0.05; ***, P < 0.001).

FIG 3

HP-PRRSV reduces the overall SLA-I abundance in infected PAMs. (A) PAMs were either mock infected or infected with PRRSV at an MOI of 1. At the indicated times, cells were harvested and subjected to SDS-PAGE and Western blot analyses using antibodies against SLA-I-HC, β2m, N protein, and β-actin. (B) PAMs were mock infected or infected with HP-PRRSV JXwn06 or UV-irradiated JXwn06 or HB-1/3.9 at an MOI of 1 or 0.1. At 12 h postinfection, the cells were harvested and subjected to SDS-PAGE and Western blot analyses using antibodies against SLA-I-HC, β2m, N protein, calnexin, and β-actin.

FIG 4

HP-PRRSV promotes SLA-I degradation via the ubiquitin-proteasome pathway. (A) PAMs were either mock infected or infected with HP-PRRSV at an MOI of 1. At 4 h postinfection, the cells were treated with MG132 or DMSO for 8 h and then processed and subjected to Western blot analysis. (B) PRRSV- or mock-infected PAMs were treated with 5 μM MG132 for 8 h at 4 h postinfection. The cells were then lysed and subjected to immunoprecipitation (IP) with rabbit polyclonal antibodies to SLA-I-HC or anti-β2m in conjunction with protein A-Sepharose beads, respectively. The proteins bound to the beads were separated by SDS-PAGE, transferred onto a PVDF membrane, and subjected to immunoblotting (IB) with antibodies to SLA-I-HC, β2m, or ubiquitin (Ub). (C) Mock- or PRRSV-infected PAMs were treated with Z-VAD-FMK (200 μM) or DMSO for 12 h. At 12 h postinfection, the cells were harvested and subjected to SDS-PAGE and Western blot analysis with the proper antibodies.

FIG 5

Induction of SLA-I degradation by the replicase protein nsp1α. The PAM cell line 3D4/21 (A, left, and B) and PK-15 cells (A, right) were transduced with lentiviruses expressing GFP-nsp1α, GFP, or other nsp's (B). At 48 h postransduction, cells were harvested and subjected to SDS-PAGE and Western blot analysis with antibodies to SLA-I-HC, β2m, GFP, and β-actin. The data are representative of results from three independent experiments.

FIG 6

Structural integrity of nsp1α is required to induce SLA-I-HC and β2m degradation. (A) Summary of the constructs used in this study. (B and C) HEK 293T cells were transfected to express FLAG–SLA-I-HC (B) or Myc-β2m (C) together with either HA-nsp1α or its truncation mutants. At 36 h posttransfection, the cells were lysed and subjected to SDS-PAGE and Western blot analysis with antibodies against FLAG, HA, and β-actin. The data are representative of results from three independent experiments. PCP, papain-like cystine protease; N-ZF, N-terminal zinc finger; C-ZF, C-terminal zinc finger; C, cysteine; H, histidine; M, methionine.

FIG 7

nsp1α interacts with both chains of SLA-I and promotes their degradation through the ubiquitin-proteasome pathway. (A) Summary of the nsp1α constructs used in this study. (B to D) HEK 293T cells transfected with the plasmid expressing FLAG-SLA-I-HC (B) or Myc-β2m (C and D) together with the plasmid expressing either HA-nsp1α or its derivatives. At 24 h posttransfection, the cells were treated with 5 μM MG132 or DMSO for 12 h. At 36 h posttransfection, the cells were either subjected to direct Western blot analyses (B, left, C, and D, left) or lysed and immunoprecipitated with anti-FLAG (B) or Myc (D) antibodies. The proteins bound to Sepharose beads were separated by SDS-PAGE, transferred onto a PVDF membrane, and probed with the antibodies to FLAG, HA, or ubiquitin (right). β-Actin served as a loading control. The data are representative of results from three independent experiments.

FIG 8

Analysis of colocalization of nsp1α with SLA-I-HC or β2m in mammalian cells. Vero cells grown on coverslips in six-well plates were transfected at 60 to 70% confluence to express the indicated proteins, either alone (A) or pairwise (B and C). At 18 to 24 h posttransfection, cells were fixed, permeabilized, stained with the appropriate antibodies, and examined by confocal microscopy. For double transfections, the merged images are shown at the right.