Mechanism Of Downregulation Of Pulmonary Alveolar Macrophages SLA â…  Molecules By PRRSV

Major histocompatibility complex (MHC) Ⅰ molecules are important to present viral antigens and subsequently activate the cytotoxic T lymphocytes (CTL) that eliminate the virus-infected cells. Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to down-regulate the surface leukocyte antigen class Ⅰ (SLA Ⅰ) levels of monocytes/macrophages. In the present study, the mechanism related to SLA Ⅰ downregulation induced by PRRSV was analyzed in order to provide scientific evidence for understanding immunosuppression and immune invasion of PRRSV.Flow Cytometry analyses showed that the both highly pathogenic PRRSV (HP-PRRSV) JXwn06 and low pathogenic PRRSV (LP-PRRSV) HB-1/3.9 could down-regulated SLA Ⅰ levels of porcine pulmonary alveolar macrophages (PAMs), and JXwn06 exhibited the stronger ability. By measuring the kinetics of SLA Ⅰ surface internalization and SLA Ⅰ export of PAMs, the results showed that PRRSV could decrease the level of SLA Ⅰ export to cell surface, which contributed to the PRRSV-mediated downregulation of SLA Ⅰ levels of PAMs, while it did not affect the kinetics of SLA Ⅰ surface internalization. Moreover, the abundances of SLA Ⅰ heavy chain (HC) and β2 microglobulin (β2m) of PAMs were shown to be reduced following PRRSV infection by western blot with the antibodies against SLA Ⅰ-HC and β2m, respectively. With the treatment of the proteasome inhibitor MG132, the SLA Ⅰ-HC and β2m abundances were shown to be increased. Additionally, compared to mock group, clear high-molecular-weight smears of the SLA Ⅰ-HC and β2m were detected in PRRSV-infected PAMs by western blot, indicating that PRRSV infection could induce the ubiquitination degradation of SLA Ⅰ-HC and β2m. These results suggest that PRRSV can induce the degradations of both SLA Ⅰ-HC and β2m through ubiquitin proteasome pathway, which contributes to the decreased level of SLA Ⅰ export to cell surface, resulting in PRRSV-mediated downregulation of SLA Ⅰ levels of PAMs.To screen which protein of PRRSV is involved in surface SLA Ⅰ downregulation,3D4/21 cells were transduced with lentiviruses that were expressing viral proteins tagged with GFP. Compared to GFP-expressing cells, the obvious decrease of surface SLA Ⅰ level was observed only in Nspla-GFP-expressing cells, and did in PK-15 and IPEC-J2 cells. Meanwhile, the SLA Ⅰ-HC and β2m abundances were also reduced in these porcine cell lines that were expressing Nspla. To investigate the molecular mechanism by which Nspla down-regulated the SLA Ⅰ-HC and β2m, the plasmids that were expressing SLA Ⅰ-HC and β2m, Nspla and two mutants of Nspla [Nsp1α (M-NZF) and Nsp1α (M-PCPa)], were constructed. The two mutants had the destructed correct conformation of N-terminal zinc finger and the activity of the papain like cysteine protease-α domain (PCPα), respectively. HEK 293T cells were co-transfected with the plasmids, and the degradation and ubiquitination assay, as well as the co-immunoprecipitation assay was then performed. The results indicated that Nspla could induce the degradations of both exogenous SLA Ⅰ-HC and β2m through ubiquitin proteasome pathway and correct confirmation of the ZF-N was required for the Nsp1α-mediated polyubiquitination of SLA Ⅰ -HC and β2m. In addition, the interactions of SLA Ⅰ-HC and β2m with Nsp1α or the Nspla mutants were detected. The results suggest that the mutations in PCPα and the ZF-N domains had no influence on the interactions between SLA Ⅰ-HC or β2m and Nspla. Moreover, Nspla was shown to be co-localized with endogenous SLA Ⅰ-HC and β2m mostly in the cytoplasm by confocal immunofluorescence assay. Additional analyses with truncated Nspla molecules demonstrated that truncated Nspla molecules lacked the ability to induce the degradations of both SLA Ⅰ-HC and β2m, indicating that structural integrity of Nsp1α is important for the function of Nsp1α to induce SLA Ⅰ-HC and β2m degradations. Taken together, our results suggest that the ZF-N is critical for the whole Nsp1α molecule to induce the degradations of both SLA Ⅰ-HC and β2m by ubiquitin proteasome pathway.In summary, our studies showed that PRRSV could induce the degradation of both SLA Ⅰ-HC and β2m through ubiquitin proteasome pathway, leading to the decreased level of SLA Ⅰ export, and resulting in PRRSV-mediated downregulation of SLA Ⅰ levels of PAMs; PRRSV Nsp1α participated in this degradations of both SLA Ⅰ-HC and β2m, sharing stronger ability to down-regulate SLA Ⅰ levels of PAMs. Our results indicate a molecular mechanism affecting antigen presentation of PAMs by PRRSV infection, and provide valuable evidence for further elucidating the mechanisms of immunosuppression and immune invasion by PRRSV.