Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the ␤2-microglobulin (␤2M) gene (B2M) and reduced cellular levels of ␤2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of ␤2M expression. Exogenous expression of Nsp4 downregulated ␤2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M. These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4.IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.KEYWORDS ␤2-microglobulin, Nsp4, porcine reproductive and respiratory syndrome virus, PRRSV, SLA-I, porcine B2M promoter M ajor histocompatibility complex class I (MHC-I) antigens in pigs are termed swine leukocyte antigen class I (SLA-I), which comprises a heterotrimeric complex consisting of the SLA-I heavy chain (SLA-I HC), ␤2-microglobulin (␤2M), and a variable peptide. In pigs, SLA-I is expressed on the surfaces of all nucleated cells to display peptide fragments derived from invading viruses for T cell recognition. This antigen presentation pathway is essential for the immune system to recognize viral peptides and initiate appropriate immune responses against invading viruses (1, 2).

Proteomic Analysis of the Secretome of Porcine Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

“…Several signaling pathways, including the TLR signaling pathways and RIG‐I‐like receptor signaling pathway, are targeted by PRRSV, suggesting a complicated antagonism between the host and PRRSV. In addition, GO analysis revealed that the down‐regulated proteins were enriched in the GO term “antigen processing and presentation.” It is known that PRRSV infection impairs expression of swine leukocyte antigen complex on the cell surface, thereby modulating antigen presentation . We observed altered expression levels of MHC class II antigen, dynactin, and TAP2, which are involved in the antigen processing and presentation pathways, suggesting that PRRSV might utilize the secreted proteins to interference with the antigen processing and presentation processes through cell‐to‐cell communications.…”

“…Highly pathogenic PRRSV strain SH‐PRRS01 used in this study was propagated in PAMs and titered by 50% cell culture infectious dose (TCID 50 ) assay in Marc‐145 cells. PAMs were isolated from 30‐day‐old clinically healthy crossbred pigs raised in the animal facility of Shanghai Academy of Agricultural Sciences, Shanghai, China, as previously described . All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Veterinary Research Institute (IACUC No: Shvri‐Pi‐0124) and performed in compliance with the Guidelines on the Humane Treatment of Laboratory Animals (Ministry of Science and Technology of the People's Republic of China, Policy No.…”

“…PRRSV was purified as previously described . To monitor PRRSV replication, PRRSV Nsp2 mRNA was detected by qRT–PCR, as previously described . The housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase was used as an internal control.…”

Molecular and Cellular Mechanisms for PRRSV Pathogenesis and Host Response to Infection

“…HP-PRRSV nsp4 has greater inhibitory effect on IFNβ than nsp4 of less pathogenic strains. As discussed above, nsp1α binds to and degrades SLA-I (Du et al, 2016a), A recent study supports this finding that HP-PRRSV nsp4 also downregulates the cellular level of β2microglobulin (β2M) (Qi et al, 2017), which forms a heterotrimeric complex with the SLA-I heavy chain and plays a critica1 role in SLA-I antigen presentation. PRRSV nsp4 binds to the B2M promoter and suppresses β2M transcription.…”

Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

“…PRRSV nsp4 performs as a 3C-like main protease that has multiple functions in the virus life cycle (65)(66)(67)(68). It plays important roles in antagonizing host antiviral immunity and in the maturation of replicase polyproteins pp1a and pp1ab (65)(66)(67)(68)(69)(70). According to EAV replicase processing, PRRSV nsp4 was supposed to mediate 9 cleavages in the nsp3-to-nsp12 region (69,70).…”