Borovec S V, Anderson D A
Hepatitis Research Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Victoria, Australia.
J Virol. 1993 Jun;67(6):3095-102. doi: 10.1128/JVI.67.6.3095-3102.1993.
To determine the mechanism for the delayed and inefficient replication of the picornavirus hepatitis A virus in cell culture, we studied the kinetics of synthesis and assembly of virus-specific proteins by metabolic labeling of infected BS-C-1 cells with L-[35S]methionine and L-[35S]cysteine. Sedimentation, electrophoresis, and autoradiography revealed the presence of virions, provirions, procapsids, and 14S (pentameric) subunits. Virions and provirions contained VP1, VP0, VP2, and VP3; procapsids contained VP1, VP0, and VP3; and pentamers contained PX, VP0, and VP3, as previously shown by immunoblotting (D.A. Anderson and B.C. Ross, J. Virol. 64:5284-5289, 1990). Under single-cycle growth conditions label was found in 14S subunits immediately after labeling from 15 to 18 h postinfection (p.i.); however, a proportion of labeled polyprotein was not cleaved and assembled into pentamers for a further 18 h. When analyzed at 72 h p.i., incorporation of label which flowed into virions was detected from 3 h p.i., with maximal uptake levels being observed from 12 to 15 h p.i. Viral antigen, infectious virus, and viral RNA were determined in parallel, with coincident peaks in these variables being observed 12 h after the period of maximum label uptake. It was also found that the lag between the synthesis of the viral polyprotein and assembly of viral particles was the same after labeling from either 12 to 15 or 27 to 30 h p.i. despite increased levels of viral RNA during this period, suggesting that factors additional to the level of RNA are involved in the restriction of viral replication. Sedimentation and immunoblot analysis revealed an additional protein of approximately 100 kDa containing both VP1- and VP2-reactive sequences and sedimenting slightly more slowly than 14S pentamers, which may represent intact P12A assembled into pentamers as has been reported for the P1 of some other picornaviruses (S. McGregor and R. R. Rueckert, J. Virol. 21:548-553, 1977). The results of this study suggest that cleavage of the hepatitis A virus polyprotein to produce pentamers is protracted (though not rate limiting) early in infection, while the assembly of pentamers into higher structures is a rapid process once sufficient viral RNA is produced for encapsidation.
为了确定微小核糖核酸病毒甲型肝炎病毒在细胞培养中复制延迟且效率低下的机制，我们通过用L-[35S]甲硫氨酸和L-[35S]半胱氨酸对受感染的BS-C-1细胞进行代谢标记，研究了病毒特异性蛋白的合成和组装动力学。沉降、电泳和放射自显影显示存在病毒体、前病毒体、原衣壳和14S（五聚体）亚基。病毒体和前病毒体含有VP1、VP0、VP2和VP3；原衣壳含有VP1、VP0和VP3；五聚体含有PX、VP0和VP3，如先前免疫印迹所示（D.A.安德森和B.C.罗斯，《病毒学杂志》64:5284 - 5289，1990）。在单循环生长条件下，感染后15至18小时（p.i.）标记后立即在14S亚基中发现标记；然而，一部分标记的多蛋白在接下来的18小时内未被切割并组装成五聚体。在感染后72小时分析时，从感染后3小时开始检测到流入病毒体的标记掺入，在感染后12至15小时观察到最大摄取水平。同时测定病毒抗原、感染性病毒和病毒RNA，在最大标记摄取期后的12小时观察到这些变量的重合峰值。还发现，无论在感染后12至15小时还是27至30小时标记，病毒多蛋白合成与病毒颗粒组装之间的延迟是相同的，尽管在此期间病毒RNA水平有所增加，这表明除了RNA水平之外，还有其他因素参与病毒复制的限制。沉降和免疫印迹分析显示存在一种约100 kDa的额外蛋白，其含有VP1和VP2反应序列且沉降速度略慢于14S五聚体，这可能代表完整的P12A组装成五聚体，正如其他一些微小核糖核酸病毒的P1所报道的那样（S.麦格雷戈和R.R.吕克特，《病毒学杂志》21:548 - 553，1977）。本研究结果表明，甲型肝炎病毒多蛋白切割产生五聚体在感染早期是延长的（尽管不是限速的），而一旦产生足够的病毒RNA用于衣壳化，五聚体组装成更高结构是一个快速过程。
