Yeh Hsiao-Yun, Yates Marylynn V, Mulchandani Ashok, Chen Wilfred
Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA.
Proc Natl Acad Sci U S A. 2008 Nov 11;105(45):17522-5. doi: 10.1073/pnas.0807066105. Epub 2008 Nov 6.
In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 replication in living Buffalo green monkey kidney (BGMK) cells via Tat peptide delivery. A nuclease-resistant MB containing 2'-O-methyl RNA bases with phosphorothioate internucleotide linkages was designed to specifically target an 18-bp 5' noncoding region of the viral genome. For intracellular delivery, a cell-penetrating Tat peptide was conjugated to the MB by using a thiol-maleimide linkage. Presence of the Tat peptide enabled nearly 100% intracellular delivery within 15 min. When the conjugate was introduced into BGMK cell monolayers infected with coxsackievirus B6, a discernible fluorescence was observed at 30 min after infection, and as few as 1 infectious viral particle could be detected within 2 h. The stability and the intracellular delivery properties of the modified MBs enabled real-time monitoring of the cell-to-cell spreading of viral infection. These results suggest that the Tat-modified, nuclease-resistant MBs may be powerful tools for improving our understanding of the dynamic behavior of viral replication and for therapeutic studies of antiviral treatments.
在本研究中，我们描述了使用抗核酸酶分子信标（MBs）通过Tat肽递送实时检测活的水牛绿猴肾（BGMK）细胞中柯萨奇病毒B6的复制情况。设计了一种含有2'-O-甲基核糖碱基和硫代磷酸酯核苷酸间连接的抗核酸酶MB，以特异性靶向病毒基因组18bp的5'非编码区。为了进行细胞内递送，使用硫醇-马来酰亚胺连接将细胞穿透性Tat肽与MB偶联。Tat肽的存在使细胞在15分钟内实现近100%的细胞内递送。当将偶联物引入感染柯萨奇病毒B6的BGMK细胞单层时，感染后30分钟观察到明显的荧光，并且在2小时内可检测到低至1个感染性病毒颗粒。修饰后的MBs的稳定性和细胞内递送特性能够实时监测病毒感染的细胞间传播。这些结果表明，Tat修饰的抗核酸酶MBs可能是用于增进我们对病毒复制动态行为的理解以及抗病毒治疗研究的有力工具。
