Visualization Of Poliovirus Plus-strand RNA In Living Host Cells

To monitor the localization and movement of the viral nucleic acids in host cells is of great importance for understanding the virus-host interaction. So far, however, methodology for such purpose is still a big challenge, especially for elucidating the intracellular dynamic mechanisms of the viral nucleic acids in living host cells. By using molecular beacon and poliovirus-Vero cell as the model virus-cell system, we established a protocol for the direct visualization of the viral nucleic acid in living host cells. After the target-specific molecular beacon was introduced into living poliovirus-infected Vero cells, the endogenous poliovirus plus-strand RNA could be clearly highlighted and its localization and mobility in living cells could be detected under a fluorescence microscope. The study verified that the target-specific molecular beacon is a powerful tool to light viral RNAs of interest in living host cells. By using living cell imaging, time-lapse monitoring and FRAP technique, the intracellular behavior of poliovirus plus-strand RNA was studied in living host cells. Poliovirus plus-strand RNA was observed to display different distribution patterns in living Vero cells at different post-infection time. Real-time monitoring showed that the distribution change for poliovirus plus-strand RNA is a characteristic translocation process that is defined as "rearrangement"since it is similar to the membrane rearrangement in poliovirus-infected cells. The translocation and rearrangement of poliovirus plus-strand RNA requires intact microtubule network of the host cell. Disruption of microtubules could alter the characteristic distribution patterns of poliovirus plus-strand RNA. Confocal-FRAP measurements showed that 49.4 ±3.2% of the poliovirus plus-strand RNA molecules diffused freely (with a D value of 9.6 ±1.6×10^-10 cm²/s) within their distribution region and the rest 50.5 ±2.9% were almost immobile and only moved very slowly with the change of RNA distribution region. The study in living host cells reveals an entrapment and diffusion mechanism for the distribution and mobility of poliovirus plus-strand RNA and demonstrates that the mechanism is mainly associated with microtubules and virus-induced membrane structures. This dynamic behavior study of viral nucleic acids in living mammalian cells brings us a deeper comprehension for the interaction of the poliovirus and its host cell. Under the electron microscope it was found that virus-induced membrane rearrangement is also microtubule-associated in poliovirus-infected Vero cells. Combined with previous reports and our findings, it is supposed that there might be a specific microtubule rearrangement accompanying the membrane rearrangement induced by poliovirus.