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Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.

Chou YY, Heaton NS, Gao Q, Palese P, Singer RH, Singer R, Lionnet T - PLoS Pathog. (2013)

Bottom Line: We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus.Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs.In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.

ABSTRACT
The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

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Colocalization of the PB2 and NA vRNAs in PR8 virus infected cells at different time points post infection.MDCK cells were infected with PR8 virus at MOI = 5 and fixed at different time points post infection. Two-color FISH was then performed using Cy5 labeled probes against the PB2 vRNAs and Cy3 labeled probes against the NA vRNAs. (A) Fluorescent images representing the PB2 vRNA (Cy5), NA vRNA (Cy3) and nuclei (DAPI). Typical images taken at 2, 4, 7, 10 and 12 hpi are presented in each lane (Maximum Intensity Projections). Scale bar = 10 µm. Zoomed-in views of the boxed regions are shown in the rightmost panel of each lane. Scale bar = 5 µm. Image Contrast was adjusted to make cytoplasmic single molecules apparent. Due to their high vRNA density, some nuclei appear uniformly bright with these settings. (B) and (C) Quantification of the colocalization efficiency between PB2 and NA vRNAs in the nucleus (B) and the cytoplasm (C). The control colocalization efficiency represents the efficiency detected between Cy3 and Cy5 signals when the two differently labeled probe sets are both targeting the NA vRNA. (D) Average copy number per cell of PB2 and NA vRNAs in infected cells at different time points post infection. The average vRNA copy number per cell is calculated by dividing the sum of PB2 and NA vRNA molecules detected by the number of nuclei in the image. A total of five images containing more than 80 cells were analyzed for each time point.
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ppat-1003358-g002: Colocalization of the PB2 and NA vRNAs in PR8 virus infected cells at different time points post infection.MDCK cells were infected with PR8 virus at MOI = 5 and fixed at different time points post infection. Two-color FISH was then performed using Cy5 labeled probes against the PB2 vRNAs and Cy3 labeled probes against the NA vRNAs. (A) Fluorescent images representing the PB2 vRNA (Cy5), NA vRNA (Cy3) and nuclei (DAPI). Typical images taken at 2, 4, 7, 10 and 12 hpi are presented in each lane (Maximum Intensity Projections). Scale bar = 10 µm. Zoomed-in views of the boxed regions are shown in the rightmost panel of each lane. Scale bar = 5 µm. Image Contrast was adjusted to make cytoplasmic single molecules apparent. Due to their high vRNA density, some nuclei appear uniformly bright with these settings. (B) and (C) Quantification of the colocalization efficiency between PB2 and NA vRNAs in the nucleus (B) and the cytoplasm (C). The control colocalization efficiency represents the efficiency detected between Cy3 and Cy5 signals when the two differently labeled probe sets are both targeting the NA vRNA. (D) Average copy number per cell of PB2 and NA vRNAs in infected cells at different time points post infection. The average vRNA copy number per cell is calculated by dividing the sum of PB2 and NA vRNA molecules detected by the number of nuclei in the image. A total of five images containing more than 80 cells were analyzed for each time point.

Mentions: In order to measure the degree of colocalization between vRNAs of different identities, cells infected with viruses were fixed and hybridized using two sets of probes targeting two different vRNAs, one labeled with Cy3 and the other labeled with Cy5. The center of each spot corresponding to a vRNA molecule was then located in 3-D space using the custom spot detection algorithm. Mapping of the different colored spots revealed the distances among spots and colocalization efficiency between the two vRNA segments could then be determined (Fig. 1A). A custom designed colocalization analysis tool was developed to measure the distances between color spots and their nearest neighbor spots of a different color. This allows the quantification of the number of spots being colocalized in individual cells. To validate the system, two control experiments were performed. The positive control experiment was done using two differently labeled probe sets (Cy3 and Cy5) targeting different regions of the same vRNA. Figure 1B showed the images of NA vRNAs detected by two differently labeled probe sets. The high degree of colocalization between the Cy3 and Cy5 spots in cells infected with PR8 virus at 6 hpi demonstrated the specificity and the sensitivity of our colocalization analysis. Quantitative colocalization analysis of the images showed that 80% of the Cy3 and Cy5 spots colocalized in the cytoplasm and 70% of them colocalized in the nucleus (Fig. 1D). To exclude the possibility that the high colocalization efficiency detected could be due to high density of vRNAs, we tested the colocalization efficiency between the highly expressed cellular β-actin mRNA and NA vRNA. The MDCK cells infected with PR8 virus were hybridized with Cy3 labeled probes for β -actin mRNA and Cy5 labeled probes targeting the NA vRNA. At 8 hpi, we observed NA vRNAs in the nucleus and the cytoplasm, whereas the β -actin mRNAs were mainly in the cytoplasm. The copy number of β-actin mRNA detected in the cytoplasm was 1409±283, similar to what was detected for viral RNAs at 4–8 hour post infection (Fig. 2D). When merging the two channels, a very small degree of colocalization between the two RNA species was observed (Fig. 1C). Quantification of the colocalization between the beta-actin mRNAs and NA vRNAs also showed a low percentage of colocalization, approximately 2.5% in the nucleus and 5% in the cytoplasm (Fig. 1E). These results demonstrated the specificity and sensitivity of our quantitative colocalization analysis.


Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.

Chou YY, Heaton NS, Gao Q, Palese P, Singer RH, Singer R, Lionnet T - PLoS Pathog. (2013)

Colocalization of the PB2 and NA vRNAs in PR8 virus infected cells at different time points post infection.MDCK cells were infected with PR8 virus at MOI = 5 and fixed at different time points post infection. Two-color FISH was then performed using Cy5 labeled probes against the PB2 vRNAs and Cy3 labeled probes against the NA vRNAs. (A) Fluorescent images representing the PB2 vRNA (Cy5), NA vRNA (Cy3) and nuclei (DAPI). Typical images taken at 2, 4, 7, 10 and 12 hpi are presented in each lane (Maximum Intensity Projections). Scale bar = 10 µm. Zoomed-in views of the boxed regions are shown in the rightmost panel of each lane. Scale bar = 5 µm. Image Contrast was adjusted to make cytoplasmic single molecules apparent. Due to their high vRNA density, some nuclei appear uniformly bright with these settings. (B) and (C) Quantification of the colocalization efficiency between PB2 and NA vRNAs in the nucleus (B) and the cytoplasm (C). The control colocalization efficiency represents the efficiency detected between Cy3 and Cy5 signals when the two differently labeled probe sets are both targeting the NA vRNA. (D) Average copy number per cell of PB2 and NA vRNAs in infected cells at different time points post infection. The average vRNA copy number per cell is calculated by dividing the sum of PB2 and NA vRNA molecules detected by the number of nuclei in the image. A total of five images containing more than 80 cells were analyzed for each time point.
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Related In: Results  -  Collection

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ppat-1003358-g002: Colocalization of the PB2 and NA vRNAs in PR8 virus infected cells at different time points post infection.MDCK cells were infected with PR8 virus at MOI = 5 and fixed at different time points post infection. Two-color FISH was then performed using Cy5 labeled probes against the PB2 vRNAs and Cy3 labeled probes against the NA vRNAs. (A) Fluorescent images representing the PB2 vRNA (Cy5), NA vRNA (Cy3) and nuclei (DAPI). Typical images taken at 2, 4, 7, 10 and 12 hpi are presented in each lane (Maximum Intensity Projections). Scale bar = 10 µm. Zoomed-in views of the boxed regions are shown in the rightmost panel of each lane. Scale bar = 5 µm. Image Contrast was adjusted to make cytoplasmic single molecules apparent. Due to their high vRNA density, some nuclei appear uniformly bright with these settings. (B) and (C) Quantification of the colocalization efficiency between PB2 and NA vRNAs in the nucleus (B) and the cytoplasm (C). The control colocalization efficiency represents the efficiency detected between Cy3 and Cy5 signals when the two differently labeled probe sets are both targeting the NA vRNA. (D) Average copy number per cell of PB2 and NA vRNAs in infected cells at different time points post infection. The average vRNA copy number per cell is calculated by dividing the sum of PB2 and NA vRNA molecules detected by the number of nuclei in the image. A total of five images containing more than 80 cells were analyzed for each time point.
Mentions: In order to measure the degree of colocalization between vRNAs of different identities, cells infected with viruses were fixed and hybridized using two sets of probes targeting two different vRNAs, one labeled with Cy3 and the other labeled with Cy5. The center of each spot corresponding to a vRNA molecule was then located in 3-D space using the custom spot detection algorithm. Mapping of the different colored spots revealed the distances among spots and colocalization efficiency between the two vRNA segments could then be determined (Fig. 1A). A custom designed colocalization analysis tool was developed to measure the distances between color spots and their nearest neighbor spots of a different color. This allows the quantification of the number of spots being colocalized in individual cells. To validate the system, two control experiments were performed. The positive control experiment was done using two differently labeled probe sets (Cy3 and Cy5) targeting different regions of the same vRNA. Figure 1B showed the images of NA vRNAs detected by two differently labeled probe sets. The high degree of colocalization between the Cy3 and Cy5 spots in cells infected with PR8 virus at 6 hpi demonstrated the specificity and the sensitivity of our colocalization analysis. Quantitative colocalization analysis of the images showed that 80% of the Cy3 and Cy5 spots colocalized in the cytoplasm and 70% of them colocalized in the nucleus (Fig. 1D). To exclude the possibility that the high colocalization efficiency detected could be due to high density of vRNAs, we tested the colocalization efficiency between the highly expressed cellular β-actin mRNA and NA vRNA. The MDCK cells infected with PR8 virus were hybridized with Cy3 labeled probes for β -actin mRNA and Cy5 labeled probes targeting the NA vRNA. At 8 hpi, we observed NA vRNAs in the nucleus and the cytoplasm, whereas the β -actin mRNAs were mainly in the cytoplasm. The copy number of β-actin mRNA detected in the cytoplasm was 1409±283, similar to what was detected for viral RNAs at 4–8 hour post infection (Fig. 2D). When merging the two channels, a very small degree of colocalization between the two RNA species was observed (Fig. 1C). Quantification of the colocalization between the beta-actin mRNAs and NA vRNAs also showed a low percentage of colocalization, approximately 2.5% in the nucleus and 5% in the cytoplasm (Fig. 1E). These results demonstrated the specificity and sensitivity of our quantitative colocalization analysis.

Bottom Line: We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus.Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs.In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.

ABSTRACT
The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

Show MeSH
Related in: MedlinePlus