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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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Related in: MedlinePlus

Colocalization of PB2 and NA vRNAs during the first hour of infection.MDCK cells were incubated in medium containing DMSO, 20 mM ammonium chloride (NH4Cl), 100 µM importazole (IPZ) or 40 ng/ml leptomycin B (LMB) during PR8 virus infection at MOI = 100. Two-color FISH was performed at 20 min, 40 min and 60 min post infection with Cy5 probes targeting the PB2 vRNA and Cy3 probes targeting the NA vRNAs. (A) For each experimental condition, four Maximum Intensity Projections of the 3D fluorescent images are shown. The upper left image shows the merged image of the Cy5, Cy3 and DAPI channels. DAPI staining (blue) stains the nucleus of the cell. The enlarged images of the square area are shown on the upper right (NA, red spots), the lower left (PB2, green spots) and the lower right (merged image of the Cy3 and Cy5 signals with DAPI). Scale bar = 5 µm. (B) Colocalization efficiency of PB2 and NA in the nuclei of PR8 virus infected MDCK cells treated with different drugs at 20 min, 40 min and 60 min post infection. (C) Colocalization efficiency of PB2 and NA in the cytoplasm of PR8 virus infected MDCK cells treated with different drugs at 20, 40 and 60 min post infection. The average copy number of vRNAs (sum of PB2 and NA vRNAs) in the nucleus (D) and the cytoplasm (E) of cells treated with different drugs are shown. For each experimental condition more than 40 cells were analyzed. An unpaired student t-test was performed for each time point between experimental groups with the mock treated group. a: p value<0.05, b: p value<0.01, c: p value<0.001, d: p value<0.0001, ns: no significance.
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ppat-1003358-g003: Colocalization of PB2 and NA vRNAs during the first hour of infection.MDCK cells were incubated in medium containing DMSO, 20 mM ammonium chloride (NH4Cl), 100 µM importazole (IPZ) or 40 ng/ml leptomycin B (LMB) during PR8 virus infection at MOI = 100. Two-color FISH was performed at 20 min, 40 min and 60 min post infection with Cy5 probes targeting the PB2 vRNA and Cy3 probes targeting the NA vRNAs. (A) For each experimental condition, four Maximum Intensity Projections of the 3D fluorescent images are shown. The upper left image shows the merged image of the Cy5, Cy3 and DAPI channels. DAPI staining (blue) stains the nucleus of the cell. The enlarged images of the square area are shown on the upper right (NA, red spots), the lower left (PB2, green spots) and the lower right (merged image of the Cy3 and Cy5 signals with DAPI). Scale bar = 5 µm. (B) Colocalization efficiency of PB2 and NA in the nuclei of PR8 virus infected MDCK cells treated with different drugs at 20 min, 40 min and 60 min post infection. (C) Colocalization efficiency of PB2 and NA in the cytoplasm of PR8 virus infected MDCK cells treated with different drugs at 20, 40 and 60 min post infection. The average copy number of vRNAs (sum of PB2 and NA vRNAs) in the nucleus (D) and the cytoplasm (E) of cells treated with different drugs are shown. For each experimental condition more than 40 cells were analyzed. An unpaired student t-test was performed for each time point between experimental groups with the mock treated group. a: p value<0.05, b: p value<0.01, c: p value<0.001, d: p value<0.0001, ns: no significance.

Mentions: Influenza virus particles enter the cells through endocytosis. Acidification of the endosomes allows the viral envelope to fuse with the endosomal membrane; influx of protons into the virus particles releases the vRNPs from the matrix protein, freeing the vRNPs into the cytosol [1]. It is unclear whether the released viral RNAs stay together or travel individually to the nucleus. In order to understand this process in more detail, MDCK cells were infected with PR8 virus at MOI = 100 and the cells were treated with ammonium chloride (NH4Cl), leptomycin B (LMB) or importazole (IPZ), to disrupt different steps of influenza vRNP nuclear transport. The colocalization efficiency of PB2 and NA vRNAs was then analyzed at 20, 40 and 60 minutes post-infection. In the mock treated cells, both PB2 and NA vRNAs could be detected in the nucleus as early as 20 minutes post infection (Fig. 3A). Replication of vRNAs started around 40 minutes post infection because the average copy number of vRNAs detected in the nucleus were comparable at 20 and 40 minutes post infection, while a significant increase was detected at 60 minutes post infection (Fig. 3D). When the colocalization of PB2 and NA vRNAs were assessed during the first hour post infection, colocalization of vRNPs in the nucleus decreased over time (from approximately 30% to 5%) while colocalization of vRNPs remained around 50% in the cytoplasm (Fig. 3B&C). This shows that vRNPs imported into the nucleus became separated, and suggests that they replicated at different positions in the nucleus. While some PB2 and NA vRNAs were not colocalized in the cytoplasm at early time points post infection, it is possible that the released vRNPs depart from each other and travel individually to the nucleus or the non-colocalized vRNPs are molecules that shuttle back into the cytoplasm after nuclear import. First, to demonstrate that the colocalized PB2 and NA vRNPs detected in the cytoplasm in the early time points post infection were predominantly vRNPs from the same virion, we retained the vRNPs in the entering virions by treating the cells with 20 mM of ammonium chloride. Ammonium chloride increases the pH value in the endosomal compartments, preventing the release of vRNPs from the matrix proteins; therefore, the vRNPs were kept in the incoming virus particles. When the cells were treated with 20 mM ammonium chloride, most of the PB2 and NA vRNAs were colocalized in the cytoplasm throughout the first hour of infection (Fig. 3A); the colocalization efficiency between these two vRNA species was also significantly higher than that in the mock treated cells (Fig. 3C). These results indicated that the uncoating and fusion events of the virions took place prior to the separation of different vRNA segments and the colocalized PB2 and NA vRNAs were the ones co-packaged in the same virus particles. In comparison with mock treated cells, lower numbers of vRNA molecules were imported into the nucleus in ammonium chloride treated cells, indicating that ammonium chloride treatment prohibited nuclear import of most vRNAs (Fig. 3D). Some vRNAs were still able to escape the inhibition and enter the nucleus. Nonetheless, the nuclear PB2 and NA vRNAs detected in the ammonium chloride treated cells exhibited decreasing levels of colocalization over time and the colocalization efficiency was significantly lower than that for the cytoplasmic vRNAs (Fig. 3B&C). These data show that vRNPs become separated once they enter the nucleus and also argue that most of the colocalized PB2 and NA vRNAs detected in the cytoplasm are from the same incoming virions instead of vRNAs from different virions infecting the same cell.


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 PB2 and NA vRNAs during the first hour of infection.MDCK cells were incubated in medium containing DMSO, 20 mM ammonium chloride (NH4Cl), 100 µM importazole (IPZ) or 40 ng/ml leptomycin B (LMB) during PR8 virus infection at MOI = 100. Two-color FISH was performed at 20 min, 40 min and 60 min post infection with Cy5 probes targeting the PB2 vRNA and Cy3 probes targeting the NA vRNAs. (A) For each experimental condition, four Maximum Intensity Projections of the 3D fluorescent images are shown. The upper left image shows the merged image of the Cy5, Cy3 and DAPI channels. DAPI staining (blue) stains the nucleus of the cell. The enlarged images of the square area are shown on the upper right (NA, red spots), the lower left (PB2, green spots) and the lower right (merged image of the Cy3 and Cy5 signals with DAPI). Scale bar = 5 µm. (B) Colocalization efficiency of PB2 and NA in the nuclei of PR8 virus infected MDCK cells treated with different drugs at 20 min, 40 min and 60 min post infection. (C) Colocalization efficiency of PB2 and NA in the cytoplasm of PR8 virus infected MDCK cells treated with different drugs at 20, 40 and 60 min post infection. The average copy number of vRNAs (sum of PB2 and NA vRNAs) in the nucleus (D) and the cytoplasm (E) of cells treated with different drugs are shown. For each experimental condition more than 40 cells were analyzed. An unpaired student t-test was performed for each time point between experimental groups with the mock treated group. a: p value<0.05, b: p value<0.01, c: p value<0.001, d: p value<0.0001, ns: no significance.
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ppat-1003358-g003: Colocalization of PB2 and NA vRNAs during the first hour of infection.MDCK cells were incubated in medium containing DMSO, 20 mM ammonium chloride (NH4Cl), 100 µM importazole (IPZ) or 40 ng/ml leptomycin B (LMB) during PR8 virus infection at MOI = 100. Two-color FISH was performed at 20 min, 40 min and 60 min post infection with Cy5 probes targeting the PB2 vRNA and Cy3 probes targeting the NA vRNAs. (A) For each experimental condition, four Maximum Intensity Projections of the 3D fluorescent images are shown. The upper left image shows the merged image of the Cy5, Cy3 and DAPI channels. DAPI staining (blue) stains the nucleus of the cell. The enlarged images of the square area are shown on the upper right (NA, red spots), the lower left (PB2, green spots) and the lower right (merged image of the Cy3 and Cy5 signals with DAPI). Scale bar = 5 µm. (B) Colocalization efficiency of PB2 and NA in the nuclei of PR8 virus infected MDCK cells treated with different drugs at 20 min, 40 min and 60 min post infection. (C) Colocalization efficiency of PB2 and NA in the cytoplasm of PR8 virus infected MDCK cells treated with different drugs at 20, 40 and 60 min post infection. The average copy number of vRNAs (sum of PB2 and NA vRNAs) in the nucleus (D) and the cytoplasm (E) of cells treated with different drugs are shown. For each experimental condition more than 40 cells were analyzed. An unpaired student t-test was performed for each time point between experimental groups with the mock treated group. a: p value<0.05, b: p value<0.01, c: p value<0.001, d: p value<0.0001, ns: no significance.
Mentions: Influenza virus particles enter the cells through endocytosis. Acidification of the endosomes allows the viral envelope to fuse with the endosomal membrane; influx of protons into the virus particles releases the vRNPs from the matrix protein, freeing the vRNPs into the cytosol [1]. It is unclear whether the released viral RNAs stay together or travel individually to the nucleus. In order to understand this process in more detail, MDCK cells were infected with PR8 virus at MOI = 100 and the cells were treated with ammonium chloride (NH4Cl), leptomycin B (LMB) or importazole (IPZ), to disrupt different steps of influenza vRNP nuclear transport. The colocalization efficiency of PB2 and NA vRNAs was then analyzed at 20, 40 and 60 minutes post-infection. In the mock treated cells, both PB2 and NA vRNAs could be detected in the nucleus as early as 20 minutes post infection (Fig. 3A). Replication of vRNAs started around 40 minutes post infection because the average copy number of vRNAs detected in the nucleus were comparable at 20 and 40 minutes post infection, while a significant increase was detected at 60 minutes post infection (Fig. 3D). When the colocalization of PB2 and NA vRNAs were assessed during the first hour post infection, colocalization of vRNPs in the nucleus decreased over time (from approximately 30% to 5%) while colocalization of vRNPs remained around 50% in the cytoplasm (Fig. 3B&C). This shows that vRNPs imported into the nucleus became separated, and suggests that they replicated at different positions in the nucleus. While some PB2 and NA vRNAs were not colocalized in the cytoplasm at early time points post infection, it is possible that the released vRNPs depart from each other and travel individually to the nucleus or the non-colocalized vRNPs are molecules that shuttle back into the cytoplasm after nuclear import. First, to demonstrate that the colocalized PB2 and NA vRNPs detected in the cytoplasm in the early time points post infection were predominantly vRNPs from the same virion, we retained the vRNPs in the entering virions by treating the cells with 20 mM of ammonium chloride. Ammonium chloride increases the pH value in the endosomal compartments, preventing the release of vRNPs from the matrix proteins; therefore, the vRNPs were kept in the incoming virus particles. When the cells were treated with 20 mM ammonium chloride, most of the PB2 and NA vRNAs were colocalized in the cytoplasm throughout the first hour of infection (Fig. 3A); the colocalization efficiency between these two vRNA species was also significantly higher than that in the mock treated cells (Fig. 3C). These results indicated that the uncoating and fusion events of the virions took place prior to the separation of different vRNA segments and the colocalized PB2 and NA vRNAs were the ones co-packaged in the same virus particles. In comparison with mock treated cells, lower numbers of vRNA molecules were imported into the nucleus in ammonium chloride treated cells, indicating that ammonium chloride treatment prohibited nuclear import of most vRNAs (Fig. 3D). Some vRNAs were still able to escape the inhibition and enter the nucleus. Nonetheless, the nuclear PB2 and NA vRNAs detected in the ammonium chloride treated cells exhibited decreasing levels of colocalization over time and the colocalization efficiency was significantly lower than that for the cytoplasmic vRNAs (Fig. 3B&C). These data show that vRNPs become separated once they enter the nucleus and also argue that most of the colocalized PB2 and NA vRNAs detected in the cytoplasm are from the same incoming virions instead of vRNAs from different virions infecting the same cell.

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