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Proc Natl Acad Sci USA. One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis
[Source: Proc Natl Acad Sci USA, full text: (LINK). Abstract, edited.]
One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis
Yi-ying Chou<SUP>a</SUP>,<SUP>1</SUP>, Reza Vafabakhsh<SUP>b</SUP>,<SUP>c</SUP>,<SUP>1</SUP>, Sultan Doğanay<SUP>d</SUP>, Qinshan Gao<SUP>a</SUP>, Taekjip Ha<SUP>b</SUP>,<SUP>c</SUP>,<SUP>d</SUP>, and Peter Palese<SUP>a</SUP>,<SUP>e</SUP>,<SUP>2</SUP>
<SUP></SUP>
Author Affiliations: Departments of <SUP>a</SUP>Microbiology and <SUP>e</SUP>Medicine, Mount Sinai School of Medicine, New York, NY 10029; and <SUP>b</SUP>Howard Hughes Medical Institute, <SUP>c</SUP>Department of Physics, and <SUP>d</SUP>Center for Biophysics and Computational Biology, University of Illinois at Urbana?Champaign, Urbana, IL 61801
Contributed by Peter Palese, April 10, 2012 (sent for review February 28, 2012)
Abstract
Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.
genome packaging - single-molecule imaging - viral ribonucleoprotein complex
Footnotes
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Yi-ying Chou<SUP>a</SUP>,<SUP>1</SUP>, Reza Vafabakhsh<SUP>b</SUP>,<SUP>c</SUP>,<SUP>1</SUP>, Sultan Doğanay<SUP>d</SUP>, Qinshan Gao<SUP>a</SUP>, Taekjip Ha<SUP>b</SUP>,<SUP>c</SUP>,<SUP>d</SUP>, and Peter Palese<SUP>a</SUP>,<SUP>e</SUP>,<SUP>2</SUP>
<SUP></SUP>
Author Affiliations: Departments of <SUP>a</SUP>Microbiology and <SUP>e</SUP>Medicine, Mount Sinai School of Medicine, New York, NY 10029; and <SUP>b</SUP>Howard Hughes Medical Institute, <SUP>c</SUP>Department of Physics, and <SUP>d</SUP>Center for Biophysics and Computational Biology, University of Illinois at Urbana?Champaign, Urbana, IL 61801
Contributed by Peter Palese, April 10, 2012 (sent for review February 28, 2012)
Abstract
Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.
genome packaging - single-molecule imaging - viral ribonucleoprotein complex
Footnotes
- ↵<SUP>1</SUP>Y.-y.C. and R.V. contributed equally to this work.
- ↵<SUP>2</SUP>To whom correspondence should be addressed. E-mail: peter.palese@mssm.edu.
- Author contributions: Y.-y.C., R.V., S.D., and P.P. designed research; Y.-y.C., R.V., and S.D. performed research; R.V., Q.G., and T.H. contributed new reagents/analytic tools; Y.-y.C., R.V., and S.D. analyzed data; and Y.-y.C., R.V., T.H., and P.P. wrote the paper.
- The authors declare no conflict of interest.
- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1206069109/-/DCSupplemental.