Hamster Kidney Epithelial Cells

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication

Non-invasive imaging that preserves spatial and temporal resolution is urgently needed to track pathogens in vivo. Victorio et al. tested whether phosphorodiamidate morpholino antisense oligos coupled to cell-penetrating peptides (PPMOs), previously developed as DENV2 antivirals, could be repurposed as fluorescent in-situ probes for live-cell imaging of DENV2 infection.

They determined whether the 5'SL PPMO specifically tracked DENV2 cellular infection. DENV2-infected Vero and BHK-21 cells were incubated with both PPMOs (10 µM each, 10 min) at 48 h and 72 h post-infection and immunostained for dsRNA, a marker of viral replication intermediates. DENV replication occurs in membrane-bound vesicle packets that appear as intensely fluorescent punctae around nuclei (Fig. 1A, B). To evaluate colocalization with viral replication sites, they calculated the Manders split coefficient (tM1) between dsRNA and either PPMO. The fraction of dsRNA colocalizing with 5'SL PPMO was comparable to that with CTRL PPMO in both cell lines at both time points (Fig. 1C), indicating no selective enrichment of 5'SL PPMO in replication vesicle packets over time. This was expected given that 5'SL and CTRL PPMOs exhibited high mutual colocalization (Fig. 1D, E), exceeding their colocalization with dsRNA. Similar results were observed in ZIKV-infected cells. These findings suggest that most PPMOs either occupied the same vesicles or were trapped in endosomes, rather than entering DENV2 replication compartments.