Hernandez Raquel, Glaros Trevor, Rizzo Gabrielle, Ferreira Davis F
Department of Molecular and Structural Biology, North Carolina State University, Raleigh, USA.
U.S. Army Combat Capabilities Development Command (CCDC) Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA.
Bio Protoc. 2019 May 20;9(10):e3239. doi: 10.21769/BioProtoc.3239.
Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to determine the proteome of medically important viruses, Sindbis virus (SINV), the prototypical alphavirus was chosen for evaluation. This model system was chosen specifically because the alphaviruses contain members which are human pathogens, this virus is well defined biochemically and structurally, and grows to high titers in both vertebrate and non-vertebrate host cells. The SINV proteome was investigated using this method to determine if host proteins are specifically packaged into infectious virions. It was also of interest if the SINV proteome, when grown in multiple host cells representing vertebrate and mosquito hosts, incorporated specific host proteins from all hosts. Observation of recurrent or distinctive proteins in the virus proteome aided in the determination of proteins incorporated into the virion as opposed to those bound to the particle exterior. Mass spectrometry analysis identified the total protein content of purified virions within limits of detection. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress, identifying at least one host factor integrally involved in alphavirus replication. Key to the success of this analysis is the method of virus purification which must deliver measurably infectious virus free of high levels of contaminants. For SINV and other members of the alphavirus family, this is accomplished by isopycnic centrifugation through potassium tartrate, followed by a high salt wash.
当前的质谱（MS）方法和新仪器现在比以前的蛋白质分级分离和鉴定方法能够更准确地鉴定低丰度蛋白质。这种方法是否可用于定义含膜病毒的病毒蛋白质组，令人感兴趣。为了评估质谱法确定医学上重要病毒蛋白质组的功效，选择了原型甲病毒辛德毕斯病毒（SINV）进行评估。选择这个模型系统是因为甲病毒包含人类病原体成员，这种病毒在生化和结构上定义明确，并且在脊椎动物和非脊椎动物宿主细胞中都能生长到高滴度。使用这种方法研究了SINV蛋白质组，以确定宿主蛋白质是否被特异性包装到感染性病毒粒子中。同样令人感兴趣的是，当SINV蛋白质组在代表脊椎动物和蚊子宿主的多种宿主细胞中生长时，是否会整合来自所有宿主的特定宿主蛋白质。观察病毒蛋白质组中反复出现或独特的蛋白质有助于确定整合到病毒粒子中的蛋白质，而不是那些与粒子外部结合的蛋白质。质谱分析确定了纯化病毒粒子在检测限内的总蛋白质含量。最显著的发现是，除了宿主蛋白质外，在所有检测的宿主细胞中生长的病毒粒子内都检测到了SINV非结构蛋白2（nsP2）。该分析鉴定了以前与甲病毒进入、复制或释放无关的宿主因子，确定了至少一种在甲病毒复制中不可或缺的宿主因子。该分析成功的关键在于病毒纯化方法，该方法必须提供可测量的无高水平污染物的感染性病毒。对于SINV和甲病毒家族的其他成员，这是通过酒石酸钾密度梯度离心，然后进行高盐洗涤来实现的。
