Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, New York, USA.
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, New York, USA
mBio. 2020 Jun 30;11(3):e01382-20. doi: 10.1128/mBio.01382-20.
Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor. The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified , but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies.
甲病毒是有包膜的正链 RNA 病毒，可引起关节炎和脑炎等严重人类疾病。尽管它们分布广泛且具有重要的医学意义，但目前尚无针对甲病毒感染的许可疫苗或抗病毒药物。盐酸小檗碱 (BBC) 是一种广谱甲病毒抑制剂，先前在基于复制子的小分子筛选中被发现。这项工作表明 BBC 抑制甲病毒复制，但也表明 BBC 可能在病毒生命周期的后期具有其他作用。在这里，我们表明 BBC 具有针对病毒核衣壳 (NC) 核心的晚期作用。在感染后期用 BBC 处理感染的细胞，无法形成稳定的细胞质 NC 或组装中间体，如通过梯度沉淀测定。使用重组衣壳蛋白 (Cp) 和纯化的基因组 RNA (gRNA) 的研究表明，BBC 扰乱了类似核心的粒子形成，并可能使组装过程陷入中间状态。尽管产生了有效的粒子并仅轻微降低了基因组包装，但来自 BBC 处理细胞的粒子的感染力降低。此外，BBC 处理游离病毒粒子强烈降低了甲病毒的感染力。相比之下，负链 RNA 病毒水疱性口炎病毒的感染力对 BBC 处理感染细胞或游离病毒的抵抗力。总之，我们的数据表明 BBC 改变了甲病毒 Cp-gRNA 相互作用和寡聚化，并表明这可能导致 NC 组装缺陷和随后的病毒进入过程中的解体缺陷。因此，BBC 可被视为新型甲病毒 NC 组装抑制剂。甲病毒基孔肯雅病毒 (CHIKV) 是一种具有增加和快速全球传播的新兴人类病原体的示例。尽管急性 CHIKV 感染很少致命，但许多患者多年来都患有衰弱性慢性关节炎。已经鉴定出针对基孔肯雅病毒和其他甲病毒的抗病毒药物，但迄今为止尚未显示其有效且已获准用于人类使用。在这里，我们研究了一种小分子，盐酸小檗碱 (BBC)，并表明它抑制了包括 CHIKV 在内的几种甲病毒的感染性病毒产生。BBC 作用于甲病毒出口途径的晚期步骤，即形成包含传染性病毒 RNA 的核衣壳。更好地了解核衣壳的形成及其被 BBC 抑制将为传染性甲病毒产生的机制提供重要信息，并可能使其在抗病毒策略中的未来靶向成为可能。
