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Chikungunya virus (CHIKV) is a reemerging alphavirus responsible for large-scale outbreaks in tropical regions. Its RNA replication depends on the assembly of functional replication complexes using the P123 and P1234 polyprotein precursors and their cleavage products, the nonstructural proteins (nsP1–nsP4). To dissect this process, we developed a trans-complementation assay using either plasmid-based expression or tetracycline-inducible stable cell lines expressing individual nsPs to rescue the activities of defective replicases. CHIKV nsP1, as well as nsP1 from closely related alphaviruses such as Ross River virus, successfully complemented CHIKV replicases carrying RNA capping-deficient mutations in nsP1. However, no complementation was observed for a replicase with an nsP1 mutation that completely disrupted membrane association. CHIKV and Eastern equine encephalitis virus (EEEV) nsP4 formed functional replication complexes with matching P123, as well as with P123 from most of alphaviruses. Genomes of CHIKV and EEEV lacking the nsP4 region remained infectious in cells expressing the corresponding nsP4 and could be propagated under these conditions. CHIKV replicase containing a mutation in the protease active site of nsP2 was also rescued by transient expression of wild-type nsP2. In contrast, replicases with mutations in the active site of the NTPase/RTPase/helicase domain of nsP2, or in nsP3 affecting phosphorylation or ADP-ribose binding/hydrolysis, could not be complemented. These results reveal key functional interdependencies among CHIKV nonstructural proteins. The inducible cell lines and trans-complementation platform for CHIKV and EEEV lacking nsP4 represent valuable tools for generating conditionally infectious virus systems and for facilitating high-throughput antiviral and neutralizing antibody screening under lower biosafety conditions.基孔肯雅病毒（Chikungunya virus, CHIKV）是一种再现性甲病毒，可在热带地区引发大规模暴发疫情。其RNA复制依赖于利用P123、P1234多聚蛋白前体及其切割产物——非结构蛋白（nsP1至nsP4）组装功能性复制复合物。为解析这一过程，我们开发了反式互补试验系统，可通过基于质粒的表达体系或四环素诱导型稳定细胞系分别表达各非结构蛋白，以拯救缺陷型复制酶的活性。基孔肯雅病毒的nsP1，以及罗斯河病毒（Ross River virus）等近缘甲病毒的nsP1，均可成功拯救携带nsP1区域RNA加帽缺陷突变的基孔肯雅病毒复制酶。但对于携带完全破坏膜结合能力的nsP1突变的复制酶，则未观察到互补现象。基孔肯雅病毒与东部马脑炎病毒（Eastern equine encephalitis virus, EEEV）的nsP4，可与匹配的P123以及多数甲病毒的P123组装形成功能性复制复合物。缺失nsP4区域的基孔肯雅病毒与东部马脑炎病毒基因组，在表达对应nsP4的细胞中仍保持感染性，且可在该条件下完成病毒扩增。携带nsP2蛋白酶活性位点突变的基孔肯雅病毒复制酶，也可通过野生型nsP2的瞬时表达实现拯救。与之相反，携带nsP2的NTP酶/RTP酶/解旋酶结构域活性位点突变，或携带影响磷酸化、ADP核糖结合/水解功能的nsP3突变的复制酶，则无法实现互补。上述结果揭示了基孔肯雅病毒非结构蛋白之间关键的功能互作关系。针对缺失nsP4的基孔肯雅病毒与东部马脑炎病毒所开发的诱导型细胞系及反式互补平台，可作为构建条件感染性病毒系统的宝贵工具，同时也能在较低生物安全等级条件下助力高通量抗病毒药物与中和抗体筛选工作。