Intracellular Localization of Poliovirus Plus- and Minus-Strand RNA Visualized by Strand-Specific Fluorescent In Situ Hybridization
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ABSTRACT 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 visualized by fluorescent in situ hybridization (FISH) with strand-specific 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 distinct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in smaller clusters of vesicles. Association of viral RNA with membranes was demonstrated by combining FISH with immunofluorescence (IF) detection 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-associated membranous structures migrated to the center of the cell. During this process, the plus- and minus-strand-containing larger structures stayed as recognizable entities, whereas the plus-strand-containing granules coalesced into a juxtanuclear area of membranous vesicles. An involvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF with 2C- and Golgi-specific antibodies.In recent years, the development of in situ technologies has made good progress. In situ hybridization (ISH) has become an important tool and has enabled the pathologist to demonstrate infectious pathogens or mRNAs in tissue sections or cytospins without destruction of morphology, thus enabling the assignment of signals to individual cells or cell compartments (, , , , , , , , ).
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Improved methods for both in situ hybridization and in situ enzyme histochemistry were described. The procedures for both methods have been significantly simplified by omitting some unnecessary treatments and substituting the cumbersome and laborious techniques, and the reliability of in situ histochemistry was increased by reversing the operations of Block and Debrouwer's procedure. The improved steps are: Instead of the conventional fixation, a simplified FAA procedure by adding liquid nitrogen onto the embedded tissues during sectioning to ensure high quality of the sections;labeled DNA by randompriming, other than labeled RNA by transcription, was used as probes in hybridization, which was conducted in a moisturesaturated plastic chamber other than emerging in mineral oil. The improved procedure for tissue in situ histochemical study was that the GUS coloration was carried out before fixation, embedding and sectioning, which was different from the procedure as described by Block and Debrouwer.
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Hybridization probe
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SUMMARY When Detroit cells are infected with poliovirus in the presence of 5 µg./ml. of actinomycin, cell-controlled RNA synthesis is blocked and 2-[14C]uridine is incorporated only into poliovirus-specific RNA. The addition of polyinosinic-polycytidylic acid (5 µg./ml.) under such conditions results in the inhibition of synthesis of 35 s intracellular, infectious poliovirus RNA. A high molecular weight fraction of poliovirus-specific RNA migrating on polyacrylamide gels, as would be expected of the double-stranded, replicative form of poliovirus RNA, is synthesized to the same extent as in the absence of interferon inducer. This RNA was digested with RNase and DNase and was resistant to the enzymes, confirming that the compound was replicative form poliovirus RNA. A low molecular weight, virus-induced RNA of 4s and unknown function is also synthesized independently of polyinosinic-polycytidylic acid. The significance of the differential inhibition in the synthesis of the compounds presumed to be poliovirus RNA and replicative form poliovirus RNA, and the fact that the capacity of polyinosinic-polycytidylic acid to inhibit the synthesis of infectious RNA is insensitive to actinomycin, is discussed.
Nuclease protection assay
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Cell-free translation of poliovirus RNA in an extract of uninfected human (HeLa) cells yielded viral proteins through proteolysis of the polyprotein. In the extract, newly synthesized proteins catalyzed poliovirus-specific RNA synthesis, and formed infectious poliovirus de novo. Newly formed virions were neutralized by type-specific antiserum, and infection of human cells with them was prevented by poliovirus receptor-specific antibodies. Poliovirus synthesis was increased nearly 70-fold when nucleoside triphosphates were added, but it was abolished in the presence of inhibitors of translation or viral genome replication. The ability to conduct cell-free synthesis of poliovirus will aid in the study of picornavirus proliferation and in the search for the control of picornaviral disease.
Picornavirus
HeLa
Picornaviridae
De novo synthesis
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Titers of antibody to poliovirus in 102 patients were determined with a sensitive neutralization assay before and 1 year after autologous bone marrow transplantation. At 1 year 14 patients (14%) had lost antibodies to poliovirus type 1 (P < .001), 10 (10%) to poliovirus type 2 (P < .05), and 13 (13%) to poliovirus type 3 (P < .01). Twenty-two patients had lost antibodies to at least one type of poliovirus. Follow-up of unimmunized patients 2 years (n = 40) and 3 years (n = 23) after transplantation documented a continuous decrease in antibody titer; by 3 years after transplantation, another 6 patients had become seronegative. When one dose of inactivated trivalent poliovirus vaccine was administered 1 year after transplantation, 2 (25%) of 8 patients seronegative for poliovirus type 1 had an increase of at least fourfold in antibody titer; after three doses, 10 (83%) of 12 patients exhibited such an increase (P < .05). The corresponding figures were 5 (71%) of 7 and 13 (100%) of 13 for poliovirus type 2 (difference not significantly) and 2 (22%) of 9 and 14 (88%) of 16 for poliovirus type 3 (P < .01). These results indicate that at least 30% of patients undergoing autologous bone marrow transplantation including those seronegative for poliovirus before transplantation will benefit from reimmunization with three doses of inactivated trivalent poliovirus vaccine 1 year after transplantation.
Inactivated Poliovirus Vaccine
Antibody titer
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Riboprobe
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Viral Interference
RNA Silencing
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