Arizala Jerlisa Ann C, Takahashi Mayumi, Burnett John C, Ouellet Dominique L, Li Haitang, Rossi John J
1 Department of Molecular and Cellular Biology, Beckman Research Institute at the City of Hope , Duarte, California.
2 Irell & Manella Graduate School of Biological Sciences , Duarte, California.
AIDS Res Hum Retroviruses. 2018 Nov;34(11):961-981. doi: 10.1089/AID.2017.0306. Epub 2018 Jul 5.
Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets-the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli-speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1. HIV-1 variants were capable of CD4 host entry and reverse transcription as WT HIV-1, but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations.
由于耐药性和持续潜伏，联合抗逆转录病毒疗法无法完全抑制HIV-1。新型治疗干预需要了解负责病毒复制的细胞内途径，特别是那些抗逆转录病毒药物未靶向的途径。一个研究较少的现象是Rev磷蛋白的核仁定位，它通过与内含子顺式作用靶点——Rev反应元件（RRE）多聚化，完成未剪接/部分剪接的HIV mRNA的核质运输。Rev包含一个核仁定位信号（NoLS），由富含精氨酸基序的COOH末端组成，用于在核仁内积累——推测这是Rev与介导mRNA非依赖性核输出和剪接的细胞蛋白相互作用的基础。在Rev-NoLS内的缺失和单点突变背景下，研究了HIV-1产生和感染过程中Rev核仁进入的功能。假设Rev-NoLS上诱导的突变会使HIV-1感染周期失活。由于多个精氨酸残基的缺失或替换，缺乏核仁进入的Rev突变导致HIV-1复制停止。缺失单个精氨酸残基的Rev突变在模式上仍严格定位于核仁，并参与前病毒产生，然而效率降低。核仁定位突变表达后，病毒RNA包装效率也降低。在病毒主干（M4、M5和M6）内含有核仁定位突变的变异HIV-1传播过程中观察到了这些结果。用Rev单点突变产生的慢病毒颗粒转导频率极低。同样，与强毒野生型（WT）HIV-1不同，HIV-1 Rev-NoLS变异体失去了感染性。HIV-1变异体能够像WT HIV-1一样进入CD4宿主并进行逆转录，但缺乏完成完整感染周期的能力。我们目前发现，在存在Rev-NoLS突变的情况下，病毒整合失调。
