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.

Fig. 1.

MB backbone modification and nuclease sensitivity study. (A) A schematic representation of the Tat-modified, nuclease-resistant MB. The phosphodiester bond was modified by replacing a nonbridging oxygen with sulfur and the 2′-sugar deoxy with 2′-O-methyl group. At room temperature, the thiol group at the quencher end reacted (≈2 h) with a maleimide group placed at the N terminus of the peptide to yield a chemically stable thioether bond. (B) Nuclease sensitivity assays using ribonuclease-free DNase I. The fluorescence of the nuclease-resistant MB is shown in yellow, and the fluorescence of an unmodified MB is shown in red. The background fluorescent signals (shown in black and green) without DNase I addition are also shown. (C) Kinetics of hybridization of Tat-modified MB CVB6 with (green) or without (orange) complementary oligonucleotides.

Fig. 2.

Intracellular delivery of MB CBV6-Tat–target hybrids (A) or MB without Tat modification or without targets (B). BGMK cells were incubated with 1 μM MB for 12 h, and images were captured by using a fluorescent microscope. (Scale bar, 20 μm.)

Fig. 3.

In vivo detection of CVB6 in BGMK cells. (A) Visualization of BGMK cells infected with 0, 1, or 10⁵ pfu at 2 h p.i. (B) The correlation between the number of plaque-forming units and fluorescent cells at 2 h p.i. Error bars represent the standard deviation of 3 replicate experiments. (Scale bar, 40 μm.)

Fig. 4.

Real-time detection of viral spreading. BGMK cells were first incubated with 1 μM MB, infected with CVB6 at an multiplicity of infection of 0.01 pfu/cell, and monitored by using a fluorescent microscope. (Scale bar, 20 μm.)