INTRACELLULAR-LOCALIZATION OF POLIOVIRUS PLUS-STRAND AND MINUS-STRANDRNA VISUALIZED BY STRAND-SPECIFIC FLUORESCENT IN-SITU HYBRIDIZATION

The time courses of poliovirus plus- and minus-strand RNA synthesis in infected HEp-2 cells were monitored separately, using a quantitative RNase assay, In parallel, viral RNA and proteins were located in situ by confocal microscopy within cells fixed by a protocol determined to retain their native size and shape. Plus- and minus-strand RNAs were v isualized by fluorescent in situ hybridization (FISH) with strand-spec ific riboprobes. The probes were labelled with different fluorochromes to allow for the simultaneous detection of plus- and minus-strand RNA . The FISH experiments showed minus-strand RNA to be present in distin ct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in s maller clusters of vesicles. Association of viral RNA with membranes w as demonstrated by combining FISH with immunofluorescence (IF) detecti on of the viral 2B- and 2C-containing P2 proteins, which are known to be markers for virus-induced membranes, At early times postinfection, the virus-induced membranous structures were distributed through most of the cytoplasm, whereas around peak RNA synthesis, both RNA-associat ed membranous structures migrated to the center of the cell. During th is process, the plus- and minus-strand-containing larger structures st ayed as recognizable entities, whereas the plus-strand-containing gran ules coalesced into a juxtanuclear area of membranous vesicles, An inv olvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF w ith 2C- and Golgi-specific antibodies.