Abstract:
CRISPR-Cas systems are widespread in prokaryotes and provide adaptive immune against viral infection. Viruses encode a type of proteins called anti-CRISPR to evade the immunity. Here, we identify an archaeal virus-encoded anti-CRISPR protein, AcrIIIB2, that inhibits Type III-B immunity. We find that AcrIIIB2 inhibits Type III-B CRISPR-Cas immunity in vivo regardless of viral early or middle-/late-expressed genes to be targeted. We also demonstrate that AcrIIIB2 interacts with Cmr4α subunit, forming a complex with target RNA and Cmr-α ribonucleoprotein complex (RNP). Furtherly, we discover that AcrIIIB2 inhibits the RNase activity, ssDNase activity and cOA synthesis activity of Cmr-α RNP in vitro under a higher target RNA-to-Cmr-α RNP ratio and has no effect on Cmr-α activities at the target RNA-to-Cmr-α RNP ratio of 1. Our results suggest that once the target RNA is cleaved by Cmr-α RNP, AcrIIIB2 probably inhibits the disassociation of cleaved target RNA, therefore blocking the access of other target RNA substrates. Together, our findings highlight the multiple functions of a novel anti-CRISPR protein on inhibition of the most complicated CRISPR-Cas system targeting the genes involved in the whole life cycle of viruses.
摘要:
CRISPR-Cas系统广泛存在于原核生物中并提供针对病毒感染的适应性免疫。病毒编码一种称为抗CRISPR的蛋白质来逃避免疫。这里,我们鉴定了古细菌病毒编码的抗CRISPR蛋白,AcrIIIB2,抑制III-B型免疫。我们发现AcrIIIB2在体内抑制III-B型CRISPR-Cas免疫,无论靶向的病毒早期或中期/晚期表达基因。我们还证明AcrIIIB2与Cmr4α亚基相互作用,与靶RNA和Cmr-α核糖核蛋白复合物(RNP)形成复合物。更进一步,我们发现AcrIIIB2抑制RNase活性,在较高的靶RNA与Cmr-αRNP比率下,体外Cmr-αRNP的ssDNase活性和cOA合成活性,并且在靶RNA与Cmr-αRNP比率为1时对Cmr-α活性没有影响。我们的结果表明,一旦目标RNA被Cmr-αRNP切割,AcrIIIB2可能抑制切割的靶RNA的解离,因此阻断其他靶RNA底物的进入。一起,我们的研究结果强调了一种新型抗CRISPR蛋白对抑制最复杂的CRISPR-Cas系统的多种功能,该系统靶向涉及病毒整个生命周期的基因.