title: Visualizing the dynamics of viral replication in living cells via Tat peptide delivery of nuclease-resistant molecular beacons.
abstract: 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.
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA.
authors.forename: Hsiao-Yun
authors.initials: HY
authors.lastname: Yeh
authors.forename: Marylynn V
authors.initials: MV
authors.lastname: Yates
authors.forename: Ashok
authors.initials: A
authors.lastname: Mulchandani
authors.forename: Wilfred
authors.initials: W
authors.lastname: Chen
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003384:Coxsackievirus Infections / Q000175:diagnosis* / Q000821:virology; D029822:Enterovirus B, Human / Q000502:physiology*; D019793:Fluorescein; D015696:Gene Products, tat / Q000378:metabolism*; D005819:Genetic Markers / Q000235:genetics*; D008856:Microscopy, Fluorescence; D012367:RNA, Viral / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D015696:Gene Products, tat; D005819:Genetic Markers; D012367:RNA, Viral; D019793:Fluorescein