The major structural protein of African swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum
The major structural protein of African swine fever virus, p73, is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum
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DOI：
10.1128/jvi.72.6.5215-5223.1998
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发表时间：
1998-06-01
影响因子：
5.4
通讯作者：
Wileman, T
中科院分区：
文献类型：
--
作者：
Cobbold, C;Wileman, T
非洲猪瘟病毒（African swine fever virus，ASFV）是一种具有二十面体对称性的大囊膜DNA病毒，为了解ASFV的组装机制，我们对ASFV的主要结构蛋白p73的生物合成和亚细胞分布进行了研究，从感染细胞制备的裂解物的蔗糖密度沉降显示，新合成的p73掺入到大小为150至250 kDa。通过体外翻译合成的p73在70 kDa处迁移，表明细胞和/或病毒蛋白是形成150- 250-kDa复合物所必需的。在2小时追踪期间，约50%的新合成的p73库结合到内质网（ER）。在此期间，p73的膜结合库而不是胞质库形成约50，000 kDa的大复合物。在细胞分泌的病毒粒子中也检测到含有p73的50，000-kDa复合物。蔗糖梯度与超免疫猪血清的免疫沉淀表明，p73是50，000 kDa复合物的主要成分。因此，该复合物可能含有600至700个拷贝的p73。复合物的形成和p73抑制的动力学是相似的，复合物的形成和抑制都可逆地抑制放线菌酮，表明复合物组装和ASFV抑制之间的功能联系。蛋白酶保护测定在形成病毒包膜的膜的内部和外部检测到50，000 kDa复合物。在ASFV的包膜下含有p73的复合物的鉴定表明，p73可能是ASFV的内核壳或基质的组分。外部库可以代表病毒的外部衣壳层内的p73。总之，这些数据表明，ASFV的内核基质和外衣壳的组装发生在ER膜上，并且这些结构在胞质溶胶中没有预先组装。
African swine fever virus (ASFV) is a large enveloped DNA virus that shares the striking icosahedral symmetry of iridoviruses, To understand the mechanism of assembly of ASFV, we have been studying the biosynthesis and subcellular distribution of p73, the major structural protein of ASFV, Sucrose density sedimentation of lysates prepared from infected cells showed that newly synthesized p73 was incorporated into a complex with a size of 150 to 250 kDa. p73 synthesized by in vitro translation migrated at 70 kDa, suggesting that cellular and/or viral proteins are required for the formation of the 150- to 250-kDa complex. During a 2-h chase, approximately 50% of the newly synthesized pool of p73 bound to the endoplasmic reticulum (ER), During this period, the membrane-bound pool of p73, but not the cytosolic pool, formed large complexes of approximately 50,000 kDa, The complexes were formed via assembly intermediates, and the entire membrane-associated pool of p73 was incorporated into the 50,000-kDa complex within 2 h, The 50,000-kDa complexes containing p73 were also detected in virions secreted from cells. Immunoprecipitation of sucrose gradients with sera taken from hyperimmune pigs suggested that p73 was the major component of the 50,000 kDa complex, It is possible, therefore, that the complex contains between 600 and 700 copies of p73. The kinetics of complex formation and envelopment of p73 were similar, and complex formation and envelopment were both reversibly inhibited by cycloheximide, suggesting a functional link between complex assembly and ASFV envelopment. A protease protection assay detected 50,000 kDa complexes on the inside and outside of the membranes forming the viral envelope. The identification of a complex containing p73 beneath the envelope of ASFV suggests that p73 may be a component of the inner core shell or matrix of ASFV. The outer pool may represent p73 within the outer capsid layer of the virus. In summary, the data suggest that the assembly of the inner core matrix and outer capsid of ASFV takes place on the ER membrane during envelopment and that these structures are not preassembled in the cytosol.