Chacko Nirmal, Mohanty Nihar Nalini, Biswas Sanchay Kumar, Chand Karam, Yogisharadhya Revanaiah, Pandey Awadh Bihari, Mondal Bimalendu, Shivachandra Sathish Bhadravati
Division of Virology, ICAR-Indian Veterinary Research Institute (IVRI), Regional Campus, Mukteswar, Nainital District, Uttarakhand (UK), 263138, India.
ICAR-National Institute of Veterinary Epidemiology and Disease Informatics, (NIVEDI), Bangalore, Karnataka, 560064, India.
Virus Genes. 2015 Oct;51(2):244-51. doi: 10.1007/s11262-015-1230-9. Epub 2015 Aug 30.
Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.
蓝舌病是一种由节肢动物传播的小型反刍动物非传染性出血性疾病，由蓝舌病病毒（BTV）引起。BTV编码的几种结构蛋白和非结构蛋白与致病机制有关。在本研究中，分析了蓝舌病病毒血清型的NS3蛋白序列中是否存在七肽区域和寡聚体形成。对所有26种BTV血清型的NS3序列进行生物信息学分析，发现至少存在三个卷曲螺旋基序（CCM）。在14-26位氨基酸（CCM-I）、185-198位氨基酸（CCM-II）和94-116位氨基酸（CCM-III）处鉴定出保守的α-螺旋七肽序列。其中，CCM-I靠近NS3的N端，推测参与寡聚化。此外，NS3的N端（1M-R117位氨基酸）在原核表达系统中作为重组融合蛋白过表达。重组NS3Nt蛋白的生化特性表明，它在还原或非还原条件下形成抗SDS的二聚体和高阶寡聚体（六聚体和/或八聚体）。卷曲螺旋基序被认为对NS蛋白的寡聚化至关重要，并且在由六个/八个亚基形成病毒孔蛋白环/孔中具有潜在作用，这是首次对蓝舌病病毒NS3中的CCM进行表征的研究。
