Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.

中文翻译：

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MARCH8抑制埃博拉病毒糖蛋白，人类免疫缺陷病毒1型信封糖蛋白和禽流感病毒H5N1血凝素成熟。

膜相关的RING-CH 8型（MARCH8）通过下调其细胞表面表达来强力阻止人类免疫缺陷病毒1型（HIV-1）包膜糖蛋白（Env）掺入病毒粒子，但该机制尚不清楚。我们现在报道，MARCH8还通过表面下调阻断了埃博拉病毒（EBOV）糖蛋白（GP）的掺入。为了了解这些病毒融合蛋白是如何下调的，我们通过常规的分泌途径研究了MARCH8对EBOV GP成熟和外在化的影响。当通过免疫沉淀检测到MARCH8时，它们与EBOV GP和弗林蛋白酶相互作用，当通过双分子荧光互补（BiFC）测定法追踪其位置时，GP /弗林蛋白酶复合物保留在高尔基体中。MARCH8不会降低GP表达或通过高甘露糖影响GP修饰内质网（ER）中的N-聚糖，但它抑制了高尔基体上GP上复杂N-聚糖的形成。此外，GP O-糖基化和弗林蛋白酶介导的蛋白水解裂解也受到抑制。此外，我们在EBOV GP上鉴定了一个新的弗林蛋白酶切割位点，发现只有那些完全糖基化的GP被弗林蛋白酶加工并掺入病毒体中。此外，GP的脱落和分泌均被MARCH8阻断。MARCH8还阻止弗林蛋白酶介导的HIV-1 Env（gp160）和高致病性禽流感病毒H5N1血凝素（HA）的裂解。我们的结论是，MARCH8通过禁止高尔基糖基化和蛋白水解酶裂解不同的病毒融合蛋白而具有非常广泛的抗病毒活性，这抑制了它们从高尔基体到质膜的运输和掺入病毒粒子。