Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA.
U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA.
J Virol. 2018 Jun 29;92(14). doi: 10.1128/JVI.00694-18. Print 2018 Jul 15.
Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus, to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts, and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro, and Chikungunya viruses. The most significant finding was that in addition to the host proteins, SINV nonstructural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics. Pathogenic alphaviruses, such as Chikungunya and Mayaro viruses, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens, such as dengue fever, West Nile, and Yellow fever viruses. With few exceptions, there are no vaccines or prophylactics for these agents, leaving one-third of the world population at risk of infection. Identifying effective antivirals has been a long-term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.
近年来，质谱方法和仪器的进步使得在低丰度下更准确地鉴定蛋白质成为可能。这项技术被应用于辛德比斯病毒（Sindbis virus），一种典型的甲病毒，以研究病毒蛋白质组。为了确定宿主蛋白是否被特异性地包装到甲病毒病毒粒子中，我们在多种代表脊椎动物和蚊子宿主的细胞中培养了辛德比斯病毒（SINV），并确定了纯化的病毒粒子中的总蛋白含量。这项分析鉴定了先前与甲病毒进入、复制或逸出无关的宿主因子。一种宿主蛋白，分选连接蛋白 5（Sorting nexin 5，SNX5），被证明对三种不同的甲病毒，辛德比斯病毒、马亚罗病毒和基孔肯雅病毒的复制至关重要。最重要的发现是，除了宿主蛋白外，在所有检查的宿主细胞中生长的病毒粒子中都检测到了辛德比斯病毒非结构蛋白 2（Nonstructural protein 2，nsP2）。nsP2 的蛋白和 RNA 相互作用能力及其在病毒粒子中的存在支持了 nsP2 在包装和/或新生病毒进入时的作用。尚未确定该蛋白的这一功能。总之，这项策略鉴定了至少一种宿主因子，它与甲病毒的复制密切相关。鉴定其他宿主蛋白为甲病毒在脊椎动物和无脊椎动物宿主中的复制提供了宿主相互作用的见解。这种病毒蛋白质组分析方法也可能有助于鉴定基于宿主的治疗候选蛋白。致病性甲病毒，如基孔肯雅病毒和马亚罗病毒，继续困扰着发展中国家和发达国家的公共卫生。甲病毒属于一类在自然中由吸血昆虫传播的病毒，被称为虫媒病毒（Arthropod-borne viruses）。该病毒组包含许多人类病原体，如登革热、西尼罗河和黄热病病毒。除了少数例外，这些病原体没有疫苗或预防性药物，使世界三分之一的人口面临感染风险。识别有效的抗病毒药物一直是对抗这些疾病的长期目标，不仅因为缺乏疫苗，还因为它们在正在进行的疫情中有效。基于质谱的辛德比斯病毒蛋白质组分析可以有效地鉴定参与病毒复制的宿主基因和病毒蛋白的新功能。鉴定这些因素对于预防这组病毒是非常宝贵的。
