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-Omethyl 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 Tatmodified, 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.detection ͉ infectious diseases ͉ real time

Shedding light on virus replication

“…The work by Yeh et al (1) has several ramifications. First, it provides evidence of a specific virus infection more rapidly than any presently available method.…”

“…I am pleased to comment on ''Visualizing the dynamics of viral replication in living cells via TAT peptide delivery of nuclease-resistant molecular beacons,'' by Hsiao-Yun Yeh et al (1), appearing in this issue of PNAS. I regard it as a very important contribution to animal (including human) virology because it allows observation of early events in virus replication in the host cell.…”

“…The authors state that none of these elements is really new-MBs are already used in many ways (3)(4)(5)(6), programs for probe sequence design are available on-line (1), nucleaseresistant constituents have been available for several years (1,(7)(8)(9)(10), and the TAT peptide from HIV type 1 was described years ago (11,12). What is significant here is the combination of these elements to achieve a highly desirable end.…”

Detection of Murine Norovirus-1 by Using TAT Peptide-Delivered Molecular Beacons

“…1). These highly target-specific probes produce fluorescence immediately upon binding, and they can distinguish single-nucleotide mismatches (9,15,20). Conjugation of the MB to the human immunodeficiency virus type 1 (HIV-1)-derived TAT peptide at the N terminus of the peptide using a thiol-maleimide linkage facilitates the traversing of biological membranes with nearly 100% efficiency.…”

“…Molecular beacons are increasingly used for real-time monitoring of virus replication in living cells. They have been used successfully in many in vitro hybridization assays due to their sensitivity and specificity and their ability to provide label-based and separation-free detection (5,9,15,16,19,20). Molecular beacons are single-stranded, dually labeled antisense oligonucleotide-nucleotide probes having a stemloop structure, with a fluorophore at one arm and a quencher at the other (5, 15) (Fig.…”