mBio | 兰州兽医研究所揭示PRRSV拮抗宿主抗病毒免疫反应的重要机制

近日，中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全学院肖书奇教授团队在期刊《mBio》发表题为"PRRSV N protein antagonizes host antiviral immune responses by upregulating HSPA1B to induce autophagic degradation of Fos-related antigen 1"的研究论文，研究揭示了猪繁殖与呼吸综合征病毒（PRRSV）通过上调热休克蛋白HSPA1B、诱导转录因子FRA1自噬降解进而拮抗宿主抗病毒免疫的关键分子机制，该研究为解析PRRSV免疫抑制这一防控难题提供了新的防控思路和靶点。

免疫抑制是PRRSV防控中的重大难题。PRRSV导致的严重免疫抑制使猪体更易遭受继发感染，给全球养猪业造成巨大经济损失，然而PRRSV拮抗宿主抗病毒免疫的具体分子机制尚不够明确。该研究发现，在PRRSV感染猪肺泡巨噬细胞（PRRSV感染的靶细胞）后，宿主细胞中的粒细胞-巨噬细胞集落刺激因子（CSF2）表达显著上调，且CSF2通过促进IL15表达抑制PRRSV复制；而转录因子Fos相关抗原1（FRA1）通过转录激活CSF2，增强了CSF2-IL15轴的抗病毒活性。然而，PRRSV进化出了针对宿主这一抗病毒防御反应的拮抗机制。

通过免疫共沉淀、尺寸排阻色谱、表面等离子共振等分析发现，PRRSV N蛋白直接结合分子伴侣HSPA1B与FRA1，并形成三者复合物，显著增强HSPA1B与FRA1的结合亲和力。在PRRSV各蛋白中，N蛋白是上调HSPA1B表达最显著的病毒蛋白；HSPA1B识别FRA1上的KFERQ样基序，在溶酶体相关膜蛋白LAMP2A协助下经分子伴侣自噬（CMA）将FRA1靶向溶酶体降解，从而瓦解宿主CSF2-IL15抗病毒防线。进一步研究发现，PRRSV N蛋白通过这一"劫持自噬途径拮抗免疫反应"策略，实现对宿主抗病毒防线的有效逃逸。以上结果证实，PRRSV N蛋白可通过上调热休克蛋白HSPA1B，进而诱导FRA1的自噬途径降解，从而拮抗宿主细胞CSF2-IL15的抗病毒途径。

该项研究表明，病毒劫持分子伴侣自噬抑制关键免疫调节因子，可能是PRRSV诱导机体免疫抑制的一种重要手段。该研究不仅揭示了PRRSV劫持自噬途径拮抗宿主抗病毒反应的“军备竞赛”机制，也为开发针对PRRSV感染的新型干预策略（靶向CMA途径与细胞因子稳态）提供了重要靶点。

摘要：

猪繁殖与呼吸综合征病毒（PRRSV）感染常导致猪出现严重的免疫抑制，然而，PRRSV拮抗宿主抗病毒免疫应答的机制尚不完全清楚。本研究发现，PRRSV感染猪肺泡巨噬细胞（PAMs）后，粒细胞-巨噬细胞集落刺激因子（CSF2）显著上调。CSF2上调通过促进IL15表达抑制PRRSV复制，而Fos相关抗原1（FRA1）通过转录调控增强CSF2-IL15轴的抗病毒活性。进一步研究表明，PRRSV N蛋白直接与HSPA1B相互作用，并激活HSPA1B介导的分子伴侣介导的自噬（CMA）以降解FRA1，从而拮抗宿主抗病毒免疫。本研究揭示了PRRSV N蛋白通过上调热休克蛋白HSPA1B促进FRA1自噬降解，进而抑制宿主CSF2-IL15抗病毒通路。该研究为PRRSV与宿主之间的“军备竞赛”提供了新见解，也为开发抗PRRSV感染策略提供了新视角。

研究亮点：

1. 发现宿主抗病毒新机制：PRRSV感染后宿主通过"FRA1-CSF2-IL15"细胞因子调控轴建立抗病毒防御反应来应对病毒感染。

2. 揭示PRRSV拮抗机制：PRRSV N蛋白劫持HSPA1B介导的CMA途径降解FRA1以瓦解宿主CSF2-IL15抗病毒免疫防线。

3. 揭示病毒-宿主对抗机制：刻画"宿主御敌、病毒破防"的军备竞赛，为靶向CMA与细胞因子稳态的抗PRRSV策略提供新靶点。

核心图注：

Fig. 1.CSF2 inhibits PRRSV replication.(A-B) PAMs were infected with PRRSV, followed by transcriptomic analysis. (C) MARC-145 cells were infected with PRRSV, and the mRNA expression levels of CSF1, CSF2, and CSF3 were detected at 24 hpi. (D-E) MARC-145 cells were transfected with overexpression plasmids for CSF1, CSF2, or CSF3, then infected with PRRSV, and the expression levels of the PRRSV N protein and ORF7 gene were detected at 24 hpi. (F-G) MARC-145 cells were transfected with the CSF2 overexpression plasmid, then infected with PRRSV, and the expression levels of the PRRSV N protein and ORF7 gene were detected at 24 and 36 hpi. (H-I) MARC-145 cells were infected with PRRSV, and the protein and mRNA expression levels of CSF2 were detected at 24 and 36 hpi. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the corresponding control.

Fig. 2.CSF2 upregulates IL15 to inhibit PRRSV replication.(A-B) MARC-145 cells were transfected with CSF2 siRNA or CSF2 overexpression plasmid, and the mRNA expression levels of the indicated cytokines were detected at 24 hpi. (C-E) MARC-145 cells were transfected with CSF2 siRNA or CSF2 overexpression plasmid, and the protein and mRNA expression levels of CSF2 and IL15 were detected at 24 hpi. (F-H) MARC-145 cells were transfected with IL15 overexpression plasmid or IL15 siRNA, then infected with PRRSV, and PRRSV replication levels were detected at 24 and 36 hpi. (I-J) MARC-145 cells were infected with PRRSV, and the protein and mRNA expression levels of IL15 were detected at 24 and 36 hpi. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the respective control.

Fig. 3.FRA1 activates CSF2 expression.(A-B) HEK-293T cells were transfected with CSF2 promoter truncated plasmids respectively. The dual-luciferase activity of the promoter truncated plasmids was detected 24 h after transfection. (C-D) HEK-293T cells were transfected using CSF2 promoter deletion plasmids respectively. The dual-luciferase activity of the promoter deletion plasmids was detected 24 h after transfection. The corresponding sequences of the deletion plasmid was submitted to PROMO online software to predict the possible binding transcription factors. (E-F) MARC-145 cells were transfected with the predicted transcription factor siRNA or overexpression plasmids, and the mRNA and protein expression levels of CSF2 were detected 24 h later. (G-H) MARC-145 cells were transfected with siRNA or overexpression plasmid of FRA1, and the mRNA and protein expression levels of CSF2 were detected 24 h later. *, P < 0.05; **, P< 0.01; ***, P< 0.001 compared to the respective control.

Fig. 4.FRA1 upregulates the CSF2/IL15 signaling pathway.(A) MARC-145 cells were transfected with different amounts of FRA1 overexpression plasmids, and the cells were infected with PRRSV, and the level of virus replication was measured at 24 hpi. (B-D) MARC-145 cells were transfected with siRNA or overexpression plasmid of FRA1, and the mRNA and protein expression levels of FRA1 and IL15 were detected 24 h after transfection. (E-F) MARC-145 (WT) and MARC-145-CSF2+/- (CSF2+/-) cells were transfected with FRA1 overexpression plasmids, and the expression levels of CSF2 and IL15 were detected 24 h after transfection. (G-H) MARC-145 and MARC-145-CSF2+/- cells were transfected with the FRA1 overexpression plasmid, and the expression levels of CSF2 and IL15 were detected 24 h after transfection. *, P < 0.05; **, P< 0.01; ***, P< 0.001 compared with the respective control.

Fig. 5.PRRSV induces FRA1 protein degradation.(A-D) MARC-145 and 3D4/21-CD163 cells were infected with PRRSV, and the mRNA and protein expression levels of FRA1 were detected at 24 and 36 hpi. (E) MARC-145 cells were infected with PRRSV or UV inactive PRRSV, and the protein expression level of FRA1 was detected at 24 and 36 hpi. (F-H) MARC-145 cells infected with or without PRRSV were treated with inhibitors, and FRA1 protein expression levels were measured at 24 hpi. *, P < 0.05; **, P< 0.01; ***, P< 0.001 compared with the respective control.

Fig. 6.PRRSV upregulates HSPA1B expression to degrade FRA1.(A) MARC-145 cells were transfected with vector or overexpression plasmid of autophagy-related proteins, respectively, and the protein expression level of FRA1 was detected 24 h after transfection. (B-D) MARC-145 or 3D4/21-CD163 cells were infected with PRRSV, and the protein and mRNA expression levels of HSPA1B were detected at 24 and 36 hpi. (E) The structural and non-structural protein plasmids of PRRSV were transfected into 3D4/21-CD163 cells, and the protein expression level of HSPA1B was detected 24 h after transfection. (F) MARC-145 cells were transfected with N protein plasmid, and the expression of HSPA1B protein was detected 24 and 36 h after transfection. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the corresponding control.

Fig. 7.PRRSV N protein interacts with HSPA1B and FRA1.(A-B) HSPA1B siRNA or overexpression plasmid were transfected into MARC-145 and 3D4/21-CD163 cells, and the protein expression levels of HSPA1B and FRA1 were detected 24 h after transfection. (C-D) The HSPA1B and N protein plasmid were co-transfected into HEK-293T cells, and coimmunoprecipitation and immunofluorescence detection were performed 24 h after transfection. (E-F) The FRA1 and N protein plasmid were co-transfected into HEK-293T cells, and coimmunoprecipitation and immunofluorescence detection were performed 24 h after transfection. l.

Fig. 8.PRRSV N protein enhances HSPA1B-mediated FRA1 degradation.(A) The FRA1, HSPA1B, and N protein expression plasmids were co-transfected into MARC-145 cells, and immunofluorescence detection was performed 24 h after transfection. (B) The FRA1, HSPA1B and N protein plasmid were co-transfected into HEK-293T cells, and coimmunoprecipitation detection was performed 24 h after transfection. (C) The PRRSV N, FRA1, and HSPA1B proteins were expressed using a prokaryotic expression system, then purified with a nickel column, and their purification results were examined by SDS-PAGE. (D-E) The prokaryotically expressed PRRSV N, FRA1, and HSPA1B proteins were used for SPR assays to analyze their binding interactions. (F) The Prokaryotically expressed PRRSV N, FRA1 and HSPA1B proteins were subjected to SEC analysis, and their elution profiles were examined.

Fig. 9.PRRSV N protein-mediated FRA1 degradation requires HSPA1B.(A-B) MARC-145 cells were transfected with HSPA1B siRNA or overexpression plasmid, then infected with PRRSV, and the protein expression levels of HSPA1B and FRA1 were detected at 24 hpi. (C) Amino acid mutations were introduced into the KFERQ like motif within the FRA1 protein sequence. Cells were then co transfected with either the wild type FRA1 plasmid or the FRA1 mut plasmid together with the HSPA1B plasmid, and target protein expression were subsequently detected. (D) A knockdown sequence targeting LAMP2A was synthesized and transfected into MARC 145 cells, and the mRNA and protein expression levels of LAMP2A were examined. (E) MARC 145 cells were transfected with LAMP2A siRNA and then infected with PRRSV, and the expression levels of target proteins were detected. (F) MARC 145 cells were transfected with LAMP2A siRNA and HSPA1B overexpression plasmid, and the expression levels of target proteins were detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the corresponding control.

Fig. 10.Schematic model of PRRSV antagonizing host antiviral immune response.The PRRSV N protein promotes autophagic degradation of FRA1 by upregulating HSPA1B expression, thereby antagonizing the host antiviral immune responses. The schematic was created with Biorender.com.

团队信息：

肖书奇教授为本文通讯作者，刘霄、张建武博士和博士研究生闫晓阳为本文共同第一作者。中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全学院为通讯作者单位。该研究得到国家自然科学基金、甘肃省联合基金、中国农业科学院科技创新工程等项目 的资助。

https://journals.asm.org/doi/10.1128/mbio.01091-26