Limits...
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.

Show MeSH

Related in: MedlinePlus

The colocalization of PB2 and NA vRNAs is independent of expression of viral proteins HA and M2.(A&B) MDCK cells were infected with either wild type PR8 (WT-PR8) or PR8-HA-GFP-HA viruses and fixed for two-color smFISH analysis against PB2 and NA vRNAs at 4, 6 and 8 hpi. (A) DAPI signal (blue), Cy5 fluorescence for PB2 vRNAs (green) and Cy3 fluorescence for NA vRNAs (red) for the cells infected with WT-PR8 and PR8-HA-GPF-HA viruses at 8 hpi (Maximum Intensity Projection). Scale bar = 10 µm. (B) Colocalization efficiency between PB2 and NA vRNAs in the cytoplasm of cells infected with either WT-RP8 or PR8-HA-GFP-HA viruses is shown. Error bars denote standard deviation. ns: t-test p value>0.05; no significance. (C&D) MDCK cells were infected with either PR8-WSN-M or PR8-WSN-ΔM2 at MOI = 5 and two-color FISH targeting the PB2 (Cy5 labeling) and NA (Cy3 labeling) vRNAs was performed at 6 and 8 hpi. (C) Fluorescent images of infected cells hybridized with Cy5 PB2 probes (green) and Cy3 NA probes (red) at 8 hpi (Maximum Intensity Projections). DAPI staining (blue) stains the nucleus of the cell. Scale bar = 10 µm. (D) Colocalization efficiency of PB2 and NA vRNAs in the cytoplasm of cells infected with PR8-WSN-M or PR8-WSN-ΔM2. Error bars denote standard deviations. ns: un-paired t-test no significance.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3649991&req=5

ppat-1003358-g006: The colocalization of PB2 and NA vRNAs is independent of expression of viral proteins HA and M2.(A&B) MDCK cells were infected with either wild type PR8 (WT-PR8) or PR8-HA-GFP-HA viruses and fixed for two-color smFISH analysis against PB2 and NA vRNAs at 4, 6 and 8 hpi. (A) DAPI signal (blue), Cy5 fluorescence for PB2 vRNAs (green) and Cy3 fluorescence for NA vRNAs (red) for the cells infected with WT-PR8 and PR8-HA-GPF-HA viruses at 8 hpi (Maximum Intensity Projection). Scale bar = 10 µm. (B) Colocalization efficiency between PB2 and NA vRNAs in the cytoplasm of cells infected with either WT-RP8 or PR8-HA-GFP-HA viruses is shown. Error bars denote standard deviation. ns: t-test p value>0.05; no significance. (C&D) MDCK cells were infected with either PR8-WSN-M or PR8-WSN-ΔM2 at MOI = 5 and two-color FISH targeting the PB2 (Cy5 labeling) and NA (Cy3 labeling) vRNAs was performed at 6 and 8 hpi. (C) Fluorescent images of infected cells hybridized with Cy5 PB2 probes (green) and Cy3 NA probes (red) at 8 hpi (Maximum Intensity Projections). DAPI staining (blue) stains the nucleus of the cell. Scale bar = 10 µm. (D) Colocalization efficiency of PB2 and NA vRNAs in the cytoplasm of cells infected with PR8-WSN-M or PR8-WSN-ΔM2. Error bars denote standard deviations. ns: un-paired t-test no significance.

Mentions: The viral proteins hemagglutinin and M2, specifically the cytoplasmic tail of M2, have been reported to be involved in the assembly of budding virions [21], [22]. We therefore tested whether HA and M2 are involved in the colocalization of vRNAs during their travel to the plasma membrane. A recombinant PR8 virus that lacks the HA ORF, PR8-HA-GFP-HA virus, was generated in MDCK cells which constitutively express the HA protein (MDCK-HA). The HA ORF of this virus is substituted with the ORF of the GFP gene. To test the role of HA protein in the colocalization of vRNAs, MDCK cells were infected with either the wild type PR8 (WT-PR8) or PR8-HA-GFP-HA viruses followed by smFISH and colocalization analysis of the PB2 and NA vRNAs at 4, 6 and 8 hpi. Since the PR8-HA-GFP-HA virus lacked the HA ORF, no HA protein could be encoded and the involvement of HA protein in this process can then be assessed. The PR8-HA-GFP-HA virus showed similar kinetics in vRNA replication and vRNA export as compared to the wild type PR8 virus in MDCK cells (data not shown). Comparable kinetics for the colocalization between PB2 and NA vRNAs were also observed for WT-PR8 and the PR8-HA-GFP-HA viruses (Fig. 6A&B). These results demonstrated that the expression of HA protein was not critical for the colocalization of different vRNAs in an infected 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)

The colocalization of PB2 and NA vRNAs is independent of expression of viral proteins HA and M2.(A&B) MDCK cells were infected with either wild type PR8 (WT-PR8) or PR8-HA-GFP-HA viruses and fixed for two-color smFISH analysis against PB2 and NA vRNAs at 4, 6 and 8 hpi. (A) DAPI signal (blue), Cy5 fluorescence for PB2 vRNAs (green) and Cy3 fluorescence for NA vRNAs (red) for the cells infected with WT-PR8 and PR8-HA-GPF-HA viruses at 8 hpi (Maximum Intensity Projection). Scale bar = 10 µm. (B) Colocalization efficiency between PB2 and NA vRNAs in the cytoplasm of cells infected with either WT-RP8 or PR8-HA-GFP-HA viruses is shown. Error bars denote standard deviation. ns: t-test p value>0.05; no significance. (C&D) MDCK cells were infected with either PR8-WSN-M or PR8-WSN-ΔM2 at MOI = 5 and two-color FISH targeting the PB2 (Cy5 labeling) and NA (Cy3 labeling) vRNAs was performed at 6 and 8 hpi. (C) Fluorescent images of infected cells hybridized with Cy5 PB2 probes (green) and Cy3 NA probes (red) at 8 hpi (Maximum Intensity Projections). DAPI staining (blue) stains the nucleus of the cell. Scale bar = 10 µm. (D) Colocalization efficiency of PB2 and NA vRNAs in the cytoplasm of cells infected with PR8-WSN-M or PR8-WSN-ΔM2. Error bars denote standard deviations. ns: un-paired t-test no significance.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649991&req=5

ppat-1003358-g006: The colocalization of PB2 and NA vRNAs is independent of expression of viral proteins HA and M2.(A&B) MDCK cells were infected with either wild type PR8 (WT-PR8) or PR8-HA-GFP-HA viruses and fixed for two-color smFISH analysis against PB2 and NA vRNAs at 4, 6 and 8 hpi. (A) DAPI signal (blue), Cy5 fluorescence for PB2 vRNAs (green) and Cy3 fluorescence for NA vRNAs (red) for the cells infected with WT-PR8 and PR8-HA-GPF-HA viruses at 8 hpi (Maximum Intensity Projection). Scale bar = 10 µm. (B) Colocalization efficiency between PB2 and NA vRNAs in the cytoplasm of cells infected with either WT-RP8 or PR8-HA-GFP-HA viruses is shown. Error bars denote standard deviation. ns: t-test p value>0.05; no significance. (C&D) MDCK cells were infected with either PR8-WSN-M or PR8-WSN-ΔM2 at MOI = 5 and two-color FISH targeting the PB2 (Cy5 labeling) and NA (Cy3 labeling) vRNAs was performed at 6 and 8 hpi. (C) Fluorescent images of infected cells hybridized with Cy5 PB2 probes (green) and Cy3 NA probes (red) at 8 hpi (Maximum Intensity Projections). DAPI staining (blue) stains the nucleus of the cell. Scale bar = 10 µm. (D) Colocalization efficiency of PB2 and NA vRNAs in the cytoplasm of cells infected with PR8-WSN-M or PR8-WSN-ΔM2. Error bars denote standard deviations. ns: un-paired t-test no significance.
Mentions: The viral proteins hemagglutinin and M2, specifically the cytoplasmic tail of M2, have been reported to be involved in the assembly of budding virions [21], [22]. We therefore tested whether HA and M2 are involved in the colocalization of vRNAs during their travel to the plasma membrane. A recombinant PR8 virus that lacks the HA ORF, PR8-HA-GFP-HA virus, was generated in MDCK cells which constitutively express the HA protein (MDCK-HA). The HA ORF of this virus is substituted with the ORF of the GFP gene. To test the role of HA protein in the colocalization of vRNAs, MDCK cells were infected with either the wild type PR8 (WT-PR8) or PR8-HA-GFP-HA viruses followed by smFISH and colocalization analysis of the PB2 and NA vRNAs at 4, 6 and 8 hpi. Since the PR8-HA-GFP-HA virus lacked the HA ORF, no HA protein could be encoded and the involvement of HA protein in this process can then be assessed. The PR8-HA-GFP-HA virus showed similar kinetics in vRNA replication and vRNA export as compared to the wild type PR8 virus in MDCK cells (data not shown). Comparable kinetics for the colocalization between PB2 and NA vRNAs were also observed for WT-PR8 and the PR8-HA-GFP-HA viruses (Fig. 6A&B). These results demonstrated that the expression of HA protein was not critical for the colocalization of different vRNAs in an infected 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