Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B.
Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B.
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DOI：
10.1128/spectrum.04116-22
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发表时间：
2023-02-14
影响因子：
3.7
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中科院分区：
文献类型：
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基质蛋白FPIV L结构域在多种副粘病毒中是保守的，但其功能和相关机制尚不清楚。本研究以副粘病毒纽卡斯尔病病毒（NDV）为材料，对FPIV L结构域进行了研究。拯救了两株重组NDV，每株在23 FPIV/I26 L-结构域的Phe（F23）或Pro（P24）位点携带单个氨基酸突变。仅在重组SG 10-F23 A（rSG 10-F23 A）菌株中观察到生长缺陷。随后对rSG 10-F23 A的研究表明，该菌株的毒力、致病性和复制能力均弱于野生型菌株rSG 10，并且出芽缺陷导致了这些弱点。为了揭示rSG 10-F23 A出芽缺陷的分子机制，探索了FPIV L结构域和转运（ESCRT）机制所需的内体分选复合物之间的桥接蛋白。在17个候选蛋白中，只有带电多泡体蛋白4（CHMP 4）旁系同源物被发现与NDV野生型M蛋白（M-WT）的相互作用比与突变型M蛋白（M-F23 A）的相互作用更强。M-WT的过表达，而不是M-F23 A的过表达，改变了CHMP 4的亚细胞位置的NDV出芽位点。此外，敲低CHMP 4 B（最丰富的CHMP 4蛋白）抑制rSG 10的释放，但不抑制rSG 10-F23 A的释放。从这些发现中，我们可以合理地推断FPIV L结构域的F23 A突变阻断NDV M蛋白和CHMP 4 B之间的相互作用，并且这有助于rSG 10-F23 A的出芽缺陷和随后的生长缺陷。本工作为进一步研究NDV及其它副粘病毒的FPIV L结构域奠定了基础。重要性多种病毒利用一个保守的基序，称为L-结构域，作为细胞适配器招募宿主ESCRT机器到其出芽位点。尽管20年前在一些副粘病毒中鉴定了FPIV型L结构域，但对其在病毒生命周期中的功能及其募集ESCRT机制的方法知之甚少。在本研究中，发现在23 FPV 26 L-结构域的F23位点处的单个氨基酸突变在后期阻断NDV出芽。此外，CHMP 4 B是ESCRT-III复合物的核心组分，被鉴定为将FPIV L结构域和ESCRT机制连接在一起的主要因素。这些结果扩展了以往对FPIV L结构域的认识，不仅为NDV等副粘病毒的减毒提供了新的途径，也为FPIV L结构域的进一步研究奠定了基础。
The matrix (M) protein FPIV L-domain is conserved among multiple paramyxoviruses; however, its function and the associated mechanism remain unclear. In this study, the paramyxovirus Newcastle disease virus (NDV) was employed to study the FPIV L-domain. Two recombinant NDV strains, each carrying a single amino acid mutation at the Phe (F23) or Pro (P24) site of 23FPIV/I26 L-domain, were rescued. Growth defects were observed in only the recombinant SG10-F23A (rSG10-F23A) strain. Subsequent studies focused on rSG10-F23A revealed that the virulence, pathogenicity, and replication ability of this strain were all weaker than those of wild-type strain rSG10 and that a budding deficiency contributed to those weaknesses. To uncover the molecular mechanism underlying the rSG10-F23A budding deficiency, the bridging proteins between the FPIV L-domain and endosomal sorting complex required for transported (ESCRT) machinery were explored. Among 17 candidate proteins, only the charged multivesicular body protein 4 (CHMP4) paralogues were found to interact more strongly with the NDV wild-type M protein (M-WT) than with the mutated M protein (M-F23A). Overexpression of M-WT, but not of M-F23A, changed the CHMP4 subcellular location to the NDV budding site. Furthermore, a knockdown of CHMP4B, the most abundant CHMP4 protein, inhibited the release of rSG10 but not that of rSG10-F23A. From these findings, we can reasonably infer that the F23A mutation of the FPIV L-domain blocks the interaction between the NDV M protein and CHMP4B and that this contributes to the budding deficiency and consequent growth defects of rSG10-F23A. This work lays the foundation for further study of the FPIV L-domain in NDV and other paramyxoviruses. IMPORTANCE Multiple viruses utilize a conserved motif, termed the L-domain, to act as a cellular adaptor for recruiting host ESCRT machinery to their budding site. Despite the FPIV type L-domain having been identified in some paramyxoviruses 2 decades ago, its function in virus life cycles and its method of recruiting the ESCRT machinery are poorly understood. In this study, a single amino acid mutation at the F23 site of the 23FPIV26 L-domain was found to block NDV budding at the late stage. Furthermore, CHMP4B, a core component of the ESCRT-III complex, was identified as a main factor that links the FPIV L-domain and ESCRT machinery together. These results extend previous understanding of the FPIV L-domain and, therefore, not only provide a new approach for attenuating NDV and other paramyxoviruses but also lay the foundation for further study of the FPIV L-domain.
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