Grantee Research Project Results

Fig. 3. Direct comparison of FACS and plaque assays. Confluent monolayer of BGMPV in 12-well plates were infected with PV1 (20 min absorption). For FACS assay, cells were harvested after 12 h as described, and then subjected to FACS. For plaque assay, infected cells were overlaid with 1% CMC in MEM (+ 2% FBS), incubated for 48 h, then fixed/stained with 1% crystal violet in 3.7% formaldehyde solution.

In this study, we describe the use of nuclease-resistant molecular beacons (MBs) for the real-time detection of coxsackievirus B6 (CVB6) 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 using a thiol-maleimide linkage (Fig. 4A). As expected, the modified MBs were highly resistant to nuclease cleavage by DNAase I (Fig. 4B). In contrast, an unmodified MB was susceptible to nuclease degradation, resulting in almost instantaneous increase in fluorescence. The dual modifications had no effect on the hybridization kinetics of the MB, as a rapid increase in fluorescence was observed in the presence of a complimentary target (Fig. 4C).

Fig. 4. (A) A schematic representation of the TAT-modified nuclease-resistant MB. (B) Nuclease sensitivity assays utilizing ribonuclease-free DNase I. The fluorescence of the nuclease-resistant MB is shown in yellow and the fluorescence of an un-modified 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 MBs CVB6 with (green) or without (orange) complementary oligos.

The intracellular delivery efficiency was tested by incubating 0.5, 1, or 2 μM of MB-target hybrids with a monolayer of Buffalo green monkey kidney (BGMK) cells. Fluorescence was detectable in 100% of the living cells as early as 15 min after introduction of the MB-target hybrids (Fig. 5A). The time-lapse images showed that the degree of cellular uptake increased after 15 min and reached saturation after 1 h incubation. The fluorescence intensity was constant for up to 12 h, indicating that the MB-target hybrids were retained inside the cells after delivery and remained resistant to the intracellular RNase H. Unhybridized MB was also introduced into BGMK cells and no fluorescence was detected during the same 12 h period, again indicating the intracellular resistance of the modified MBs to nuclease attack (Fig. 5B).  In contrast, in the absence of TAT, no internalization of MBs was observed and fluorescence was detected only in the medium (Fig. 5B), confirming the effectiveness of the TAT peptide for rapid intracellular delivery

Fig. 5. Intracellular delivery of (A) MB CBV6-TAT/target hybrids or (B) MB without TAT modification or without targets. BGMK cells were incubated with 1 µM MB for 12 h, and images were captured using a fluorescent microscope. Scale bar, 20 µm.

Fig. 6. (A) Visualization of BGMK cells infected with 0, 1 or 10⁵ PFU at 2 hpi (B) The correlation between the number of PFU and fluorescent cells at 2 hpi Error bars represent the standard deviation of three replicate experiments.

The ability to detect infected cells continuously should allow one to follow the spreading of infectious viruses on a real-time basis. To determine whether this was, indeed, possible, BGMK cells were infected at a very low infection dosage (MOI: 0.01 pfu/cell) and monitored continuously using a fluorescence microscope in a fixed area for 12 h. Fig. 7 shows the cell to cell progression of virus spreading at 6 representative time points. Several infected cells were observed at 15 min p.i., suggesting that the viruses entered the cells and started the uncoating process within 15 min. The number of fluorescent cells slowly increased with time suggesting continuous virus infection. By 6 h p.i., a further outward spread of fluorescent cells was observed, indicating the secondary spreading of infection from progeny virions to cells surrounding the initial infected cells. The number of fluorescent cells continued to increase with time and finally spread outward to the entire observation area by 12 h p.i. The majority of infected cells remained adherent and some fluorescence was visible outside the cells, indicating that the fluorescent hybrids with viral RNA entered the extracellular region as a result of the release of progeny virions during cell lysis.

Fig. 7. Real-time detection of viral spreading. BGMK cells were first incubated with 1 μM MB, infected with CVB6 at an M.O.I. of 0.01 pfu/cell, and monitored using a fluorescent microscope.