Downstream Sequences Control the Processing of the Pestivirus Erns-E1 Precursor
Downstream Sequences Control the Processing of the Pestivirus Erns-E1 Precursor
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DOI：
10.1128/jvi.01905-20
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发表时间：
2020-10
影响因子：
5.4
通讯作者：
Yu Mu;Ioana Bintintan;G. Meyers
中科院分区：
文献类型：
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作者：
Yu Mu;Ioana Bintintan;G. Meyers
细胞信号肽酶（SPase）裂解是病毒包膜蛋白成熟的重要步骤。该系统的微调允许在不同包膜病毒中建立协调的折叠和加工过程。我们在这里报告了瘟病毒Erns-E1-E2糖蛋白前体的SPase加工。Erns-E1切割被延迟，并且只有当完整的E1序列存在时才有效地执行。Erns-E1前体的C-末端截短损害加工并导致蛋白质的显著分泌。当引入保留E1羧基末端的内部缺失时，未检测到后者，但这些构建体也显示出受损的加工。此外，Erns-E1仅在E1/E2位点裂解后加工。因此，通过SPase对瘟病毒糖蛋白前体的加工以有序的方式进行，并且取决于蛋白质的完整性以进行有效切割。本文讨论了这种处理方案的功能重要性。与其他包膜病毒一样，瘟病毒也利用细胞内的蛋白酶来加工其结构蛋白。虽然典型的信号肽酶切割基序存在于Erns和E1/E2和E2/P7位点之前的信号序列的羧基末端，但Erns-E1前体被信号肽酶以非常不寻常的结构切割，其中切割位点上游的跨膜序列被两亲性螺旋取代。如前所示，两亲性螺旋的完整性对于有效加工至关重要。这里提供的数据表明，该切割位点下游的E1序列对于切割也是重要的。羧基末端截短的E1部分以及E1的内部缺失的切割效率降低到小于30%的野生型（wt）的水平。此外，超过30个氨基酸的C-末端截短导致未切割的融合蛋白的强烈分泌。当E1的10至5个氨基末端残基被留下时，甚至观察到加工减少和分泌增加，而通过1或3个E1残基的延伸导致加工减少，但没有显著增加分泌。与E1序列相反，与Erns C末端融合的10个氨基酸的c-myc标签对分泌只有轻微的影响，但也不能有效地加工。E2上游的von Heijne序列的突变不仅阻断了E1和E2之间的切割，而且还阻止了Erns和E2之间的加工。因此，Erns-E1位点的加工是一个高度调控的过程。细胞信号肽酶（SPase）裂解是病毒包膜蛋白成熟的重要步骤。该系统的微调允许在不同包膜病毒中建立协调的折叠和加工过程。我们在这里报告了瘟病毒Erns-E1-E2糖蛋白前体的SPase加工。Erns-E1切割被延迟，并且只有当完整的E1序列存在时才有效地执行。Erns-E1前体的C-末端截短损害加工并导致蛋白质的显著分泌。当引入保留E1羧基末端的内部缺失时，未检测到后者，但这些构建体也显示出受损的加工。此外，Erns-E1仅在E1/E2位点裂解后加工。因此，通过SPase对瘟病毒糖蛋白前体的加工以有序的方式进行，并且取决于蛋白质的完整性以进行有效切割。本文讨论了这种处理方案的功能重要性。
Cellular signal peptidase (SPase) cleavage represents an important step in maturation of viral envelope proteins. Fine tuning of this system allows for establishment of concerted folding and processing processes in different enveloped viruses. We report here on SPase processing of the Erns-E1-E2 glycoprotein precursor of pestiviruses. Erns-E1 cleavage is delayed and only executed efficiently when the complete E1 sequence is present. C-terminal truncation of the Erns-E1 precursor impairs processing and leads to significant secretion of the protein. The latter is not detected when internal deletions preserving the E1 carboxy terminus are introduced, but also these constructs show impaired processing. Moreover, Erns-E1 is only processed after cleavage at the E1/E2 site. Thus, processing of the pestiviral glycoprotein precursor by SPase is done in an ordered way and depends on the integrity of the proteins for efficient cleavage. The functional importance of this processing scheme is discussed in the paper. ABSTRACT Like other enveloped viruses, pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxy terminus of the signal sequence preceding Erns and the E1/E2 and E2/P7 sites, the Erns-E1 precursor is cleaved by signal peptidase at a highly unusual structure, in which the transmembrane sequence upstream of the cleavage site is replaced by an amphipathic helix. As shown before, the integrity of the amphipathic helix is crucial for efficient processing. The data presented here demonstrate that the E1 sequence downstream of this cleavage site is also important for the cleavage. Carboxy-terminal truncation of the E1 moiety as well as internal deletions in E1 reduced the cleavage efficiency to less than 30% of the wild-type (wt) level. Moreover, the C-terminal truncation by more than 30 amino acids resulted in strong secretion of the uncleaved fusion proteins. The reduced processing and increased secretion were even observed when 10 to 5 amino-terminal residues of E1 were left, whereas extensions by 1 or 3 E1 residues resulted in reduced processing but no significantly increased secretion. In contrast to the E1 sequences, a 10-amino-acid c-myc tag fused to the Erns C terminus had only marginal effect on secretion but was also not processed efficiently. Mutation of the von Heijne sequence upstream of E2 not only blocked the cleavage between E1 and E2 but also prevented the processing between Erns and E2. Thus, processing at the Erns-E1 site is a highly regulated process. IMPORTANCE Cellular signal peptidase (SPase) cleavage represents an important step in maturation of viral envelope proteins. Fine tuning of this system allows for establishment of concerted folding and processing processes in different enveloped viruses. We report here on SPase processing of the Erns-E1-E2 glycoprotein precursor of pestiviruses. Erns-E1 cleavage is delayed and only executed efficiently when the complete E1 sequence is present. C-terminal truncation of the Erns-E1 precursor impairs processing and leads to significant secretion of the protein. The latter is not detected when internal deletions preserving the E1 carboxy terminus are introduced, but also these constructs show impaired processing. Moreover, Erns-E1 is only processed after cleavage at the E1/E2 site. Thus, processing of the pestiviral glycoprotein precursor by SPase is done in an ordered way and depends on the integrity of the proteins for efficient cleavage. The functional importance of this processing scheme is discussed in the paper.