Ali Nida Fatima, Paracha Rehan Zafar, Tahir Muhammad
Department of Plant Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology, Islamabad, Federal, Pakistan.
Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology, Islamabad, Federal, Pakistan.
PeerJ. 2021 Oct 19;9:e12018. doi: 10.7717/peerj.12018. eCollection 2021.
Cotton leaf curl disease (CLCuD) is a disease of cotton caused by begomoviruses, leading to a drastic loss in the annual yield of the crop. Pakistan has suffered two epidemics of this disease leading to the loss of billions in annual exports. The speculation that a third epidemic of CLCuD may result as consequence of the frequent occurrence of (ToLCNDV) (CLCuKoV-Bu) in CLCuD infected samples, demand that the interactions taking between the two viruses be properly evaluated. This study is designed to assess virus-virus interactions at the molecular level and determine the type of co-infection taking place.
Based on the amino acid sequences of the gene products of both CLCuKoV-Bu and ToLCNDV, protein structures were generated using different software, , MODELLER, I-TASSER, QUARKS, LOMETS and RAPTORX. A consensus model for each protein was selected after model quality assessment using ERRAT, QMEANDisCo, PROCHECK Z-Score and Ramachandran plot analysis. The active and passive residues in the protein structures were identified using the CPORT server. Protein-Protein Docking was done using the HADDOCK webserver, and 169 Protein-Protein Interaction (PPIs) were performed between the proteins of the two viruses. The docked complexes were submitted to the PRODIGY server to identify the interacting residues between the complexes. The strongest interactions were determined based on the HADDOCK Score, Desolvation energy, Van der Waals Energy, Restraint Violation Energy, Electrostatic Energy, Buried Surface Area and Restraint Violation Energy, Binding Affinity and Dissociation constant (K). A total of 50 ns Molecular Dynamic simulations were performed on complexes that exhibited the strongest affinity in order to validate the stability of the complexes, and to remove any steric hindrances that may exist within the structures.
Our results indicate significant interactions taking place between the proteins of the two viruses. Out of all the interactions, the strongest were observed between the Replication Initiation protein (Rep) of CLCuKoV-Bu with the Movement protein (MP), Nuclear Shuttle Protein (NSP) of ToLCNDV (DNA-B), while the weakest were seen between the Replication Enhancer protein (REn) of CLCuKoV-Bu with the REn protein of ToLCNDV. The residues identified to be taking a part in interaction belonged to domains having a pivotal role in the viral life cycle and pathogenicity. It maybe deduced that the two viruses exhibit antagonistic behavior towards each other, and the type of infection may be categorised as a type of Super Infection Exclusion (SIE) or homologous interference. However, further experimentation, in the form of transient expression analysis, is needed to confirm the nature of these interactions and increase our understanding of the direct interactions taking place between two viruses.
棉花曲叶病（CLCuD）是由双生病毒引起的棉花病害，导致棉花年产量急剧下降。巴基斯坦曾遭受过两次这种病害的流行，造成每年数十亿美元的出口损失。由于在感染CLCuD的样本中频繁出现番茄褐色皱纹果病毒（ToLCNDV）和棉花曲叶古吉拉特病毒-布杰瓦尔分离物（CLCuKoV-Bu），有人猜测可能会出现第三次CLCuD流行，这就要求对这两种病毒之间的相互作用进行恰当评估。本研究旨在评估分子水平上的病毒-病毒相互作用，并确定发生的共感染类型。
基于CLCuKoV-Bu和ToLCNDV基因产物的氨基酸序列，使用不同软件，如MODELLER、I-TASSER、QUARKS、LOMETS和RAPTORX生成蛋白质结构。在使用ERRAT、QMEANDisCo、PROCHECK Z评分和拉氏图分析进行模型质量评估后，为每个蛋白质选择一个共识模型。使用CPORT服务器鉴定蛋白质结构中的活性和非活性残基。使用HADDOCK网络服务器进行蛋白质-蛋白质对接，在两种病毒的蛋白质之间进行了169次蛋白质-蛋白质相互作用（PPI）。将对接复合物提交给PRODIGY服务器，以鉴定复合物之间的相互作用残基。根据HADDOCK评分、去溶剂化能、范德华能、约束违反能、静电能、埋藏表面积和约束违反能、结合亲和力和解离常数（K）确定最强相互作用。对表现出最强亲和力的复合物进行了总共50纳秒的分子动力学模拟，以验证复合物的稳定性，并消除结构中可能存在的任何空间位阻。
我们的结果表明两种病毒的蛋白质之间发生了显著相互作用。在所有相互作用中，观察到CLCuKoV-Bu的复制起始蛋白（Rep）与ToLCNDV（DNA-B）的运动蛋白（MP）、核穿梭蛋白（NSP）之间的相互作用最强，而CLCuKoV-Bu的复制增强蛋白（REn）与ToLCNDV的REn蛋白之间的相互作用最弱。被确定参与相互作用的残基属于在病毒生命周期和致病性中起关键作用的结构域。可以推断这两种病毒对彼此表现出拮抗行为，感染类型可能归类为一种超级感染排除（SIE）或同源干扰。然而，需要以瞬时表达分析的形式进行进一步实验，以确认这些相互作用的性质，并增进我们对两种病毒之间直接相互作用的理解。
