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Defective influenza viral ribonucleoproteins cause interference
Abstract:
Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.
Insights
Defective interfering (DI) influenza virus ribonucleoproteins (RNPs) contain elevated levels of DI RNA, which is responsible for viral interference. This DI RNP fraction, distinct from infectious virus RNP, significantly reduces infectious centers.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Influenza viruses possess segmented RNA genomes packaged into ribonucleoproteins (RNPs).
- Defective interfering (DI) virus particles arise during replication and interfere with standard virus propagation.
- The precise nature and role of RNPs in DI virus interference are not fully understood.
Purpose of the Study:
- To characterize the RNP composition of infectious and DI influenza viruses.
- To investigate the interference activity associated with specific RNP fractions.
- To determine if DI RNA within RNPs is the causative agent of interference.
Main Methods:
- Glycerol gradient centrifugation to isolate RNP fractions.
- Polyacrylamide gel electrophoresis (PAGE) to analyze RNA content of RNPs.
- Infectious center reduction assay to measure interference activity.
- RNase sensitivity assays to confirm RNA's role in interference.
Main Results:
- Three major RNP peaks (I, II, III) were identified in both infectious and DI influenza viruses.
- Peak III RNP was enriched in DI virus preparations and contained characteristic DI RNA segments.
- Only Peak III RNP from DI virus exhibited significant interference activity, which was RNase sensitive.
- Interfering RNPs were heterogeneous and migrated slower than M and NS gene-containing RNPs.
Conclusions:
- DI RNA, packaged within specific DI RNPs (Peak III), is the primary agent responsible for interference.
- DI RNPs play a crucial role in the phenomenon of defective interference in segmented, negative-stranded RNA viruses.
- These findings elucidate the molecular basis of interference mediated by DI particles in influenza virus.