Conserved molecular mechanisms of replication for mosquito-borne flaviviruses

作为专性的细胞内寄生虫，所有病毒都通过病毒与宿主蛋白的相互作用来选择宿主机制进行复制。节肢动物传播的病毒通过节肢动物媒介传播给脊椎动物，必须劫持人类和节肢动物细胞的宿主机器，才能完成病毒复制的相同基本方面。因此，节肢动物传播的病毒与宿主同源物(互同物)保持蛋白质相互作用以进行复制。识别这些相互关联对于理解一组重要的病毒如何从生物物理角度处理这种独特的限制至关重要。对于由伊蚊传播的黄病毒(伊蚊传播的黄病毒)，它还可以通过扩大药物靶标清单来为治疗开发提供信息，因为这些病毒是人类疾病的主要来源。利用比较蛋白质组学方法，我们最近发现了登革病毒(DENV)相互同源的大规模证据，登革病毒是一种主要的伊蚊传播的黄病毒，每年感染近4亿人。这些相互关联涉及对病毒在人和伊蚊细胞中复制至关重要的过程。我们假设伊蚊传播的黄病毒使用保守的互补体来促进在人和伊蚊细胞中的复制，因为这些病毒受到类似的限制，并且跨多个不同宿主维持病毒-宿主蛋白相互作用的复杂性。这项建议的总体目标是系统地比较两种伊蚊传播的黄病毒在病毒复制中的相互作用。我们将重点关注DENV，以利用我们现有的相互作用数据和黄热病病毒(YFV)，这是一种重新出现的伊蚊传播的黄病毒，与DENV有遥远的亲缘关系。在目标1中，我们将使用亲和纯化和质谱法系统地鉴定YFV-人和YFV-伊蚊蛋白的相互作用。我们将通过计算网络集成进一步识别YFV相互关联。在目标2中，我们将使用类似的计算网络集成方法来识别YFV和DENV之间保守的相互关系。然后，我们将通过测量Interolog敲除后的病毒复制来测试Interolos在伊蚊传播的黄病毒复制中的作用。这项工作将确定保守的分子机制，一组医学上重要的病毒通过这些机制进行复制。它将揭示限制病毒进化的生物物理参数。在未来，我们确定的相互作用可能被用作药理或媒介工程靶标，以抑制多种伊蚊携带的黄病毒的复制。我们的工作也将为寻找更多跨不同节肢动物载体和/或不同病毒家族的相互同源奠定基础。

As obligate intracellular parasites, all viruses replicate by coopting host machinery through virus-host protein interactions. Arthropod-borne viruses, which are transmitted to vertebrates by arthropod vectors, must hijack host machinery in human and arthropod cells to accomplish the same fundamental aspects of virus replication. Thus, arthropod-borne viruses maintain protein interactions with host homologs (interologs) to replicate. Identifying these interologs is critical to understanding how an important group of viruses deals with this unique constraint from a biophysical perspective. For flaviviruses transmitted by Aedes mosquitoes (Aedes-borne flaviviruses), it can also inform therapy development by expanding the list of drug targets since these viruses are a major source of human disease. Using a comparative proteomics approach, we recently found large-scale evidence of interologs for dengue virus (DENV), a major Aedes-borne flavivirus that infects nearly 400 million people annually. These interologs involve processes that are essential for virus replication in human and Aedes cells. We hypothesize that Aedes-borne flaviviruses use the conserved interologs to facilitate replication in human and Aedes cells due to the similar constraints place on these viruses and the complexity of maintaining virus-host protein interactions across multiple divergent hosts. The overall objective of this proposal is to systematically compare the role of interologs in virus replication for two Aedes-borne flaviviruses. We will focus on DENV to take advantage of our existing interolog data and yellow fever virus (YFV), a re-emerging Aedes-borne flavivirus that is distantly related to DENV. In Aim 1, we will systematically identify YFV-human and YFV-Aedes protein interactions using affinity purification and mass spectrometry. We will further identify YFV interologs through computational network integration. In Aim 2, we will identify interologs conserved between YFV and DENV using a similar computational network integration approach. We will then test the role of interologs in Aedes-borne flavivirus replication by measuring virus replication following interolog knockdown. This work will identify the conserved molecular mechanisms by which a medically important group of viruses replicates. It will reveal the biophysical parameters that constrain virus evolution. In the future, the interologs we identify could be leveraged as pharmacological or vector engineering targets to inhibit replication of multiple Aedes-borne flaviviruses. Our work would also lay the foundation to search for more interologs conserved across different arthropod vectors and/or different virus families.