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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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Proposed model for the disassembly and colocalization of different vRNA segments in an infected cell.(1) An influenza virus particle enters the cell through the endocytic pathway. (2) Acidification of the endosome allows fusion of viral envelope with the endosomal membrane and the release of vRNPs into the cytosol. The vRNPs packaged in the virus particles remain tightly associated during their transport towards the nucleus. (3) Upon nuclear import, the associated vRNPs of different segments disassemble in the nucleus (Results in Figure 3) and (4) different vRNAs replicate in different areas of the nucleus and no segment-specific accumulation of vRNAs is observed (Results in Figure 2&3). (5) The newly synthesized vRNPs are exported into the cytoplasm individually and different segments are not associated with each other shortly after their nuclear export (Results in Figure 2&3). (6) During the vRNP trafficking to the plasma membrane, the vRNPs are loaded onto recycling endosomes through the association with Rab11 [15], [16], [17]. The vRNPs of different segments then start to colocalize with each other and travel together towards the apical plasma membrane as a pre-formed complex of eight different vRNPs. (7) As the complex reaches the plasma membrane, the eight different vRNPs are then packaged as a whole into the budding virions.
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ppat-1003358-g007: Proposed model for the disassembly and colocalization of different vRNA segments in an infected cell.(1) An influenza virus particle enters the cell through the endocytic pathway. (2) Acidification of the endosome allows fusion of viral envelope with the endosomal membrane and the release of vRNPs into the cytosol. The vRNPs packaged in the virus particles remain tightly associated during their transport towards the nucleus. (3) Upon nuclear import, the associated vRNPs of different segments disassemble in the nucleus (Results in Figure 3) and (4) different vRNAs replicate in different areas of the nucleus and no segment-specific accumulation of vRNAs is observed (Results in Figure 2&3). (5) The newly synthesized vRNPs are exported into the cytoplasm individually and different segments are not associated with each other shortly after their nuclear export (Results in Figure 2&3). (6) During the vRNP trafficking to the plasma membrane, the vRNPs are loaded onto recycling endosomes through the association with Rab11 [15], [16], [17]. The vRNPs of different segments then start to colocalize with each other and travel together towards the apical plasma membrane as a pre-formed complex of eight different vRNPs. (7) As the complex reaches the plasma membrane, the eight different vRNPs are then packaged as a whole into the budding virions.

Mentions: In this study, we have analyzed the disassembly and the subsequent assembly of influenza vRNPs segments in virus-infected cells using smFISH. We show that vRNPs of different segments remain associated after their release from the incoming virions and they travel as a package to the nuclear membrane. Newly synthesized vRNPs of different segments do not occupy the same space in the nucleus and they are likely exported individually into the cytoplasm because colocalization of the exported vRNPs of different segments is not observed during the early stages of infection. Different viral RNPs colocalize in the cytoplasm at later stages during infection (6–8 hpi in MDCK cells) and they are often found to be associated with Rab11-recycling endosomes. These results provide evidence that vRNPs belonging to different segments follow the same trafficking route and the selection for the correct combination of the eight vRNA segments likely takes place before the vRNPs reach the cell surface (Fig. 7).


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)

Proposed model for the disassembly and colocalization of different vRNA segments in an infected cell.(1) An influenza virus particle enters the cell through the endocytic pathway. (2) Acidification of the endosome allows fusion of viral envelope with the endosomal membrane and the release of vRNPs into the cytosol. The vRNPs packaged in the virus particles remain tightly associated during their transport towards the nucleus. (3) Upon nuclear import, the associated vRNPs of different segments disassemble in the nucleus (Results in Figure 3) and (4) different vRNAs replicate in different areas of the nucleus and no segment-specific accumulation of vRNAs is observed (Results in Figure 2&3). (5) The newly synthesized vRNPs are exported into the cytoplasm individually and different segments are not associated with each other shortly after their nuclear export (Results in Figure 2&3). (6) During the vRNP trafficking to the plasma membrane, the vRNPs are loaded onto recycling endosomes through the association with Rab11 [15], [16], [17]. The vRNPs of different segments then start to colocalize with each other and travel together towards the apical plasma membrane as a pre-formed complex of eight different vRNPs. (7) As the complex reaches the plasma membrane, the eight different vRNPs are then packaged as a whole into the budding virions.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649991&req=5

ppat-1003358-g007: Proposed model for the disassembly and colocalization of different vRNA segments in an infected cell.(1) An influenza virus particle enters the cell through the endocytic pathway. (2) Acidification of the endosome allows fusion of viral envelope with the endosomal membrane and the release of vRNPs into the cytosol. The vRNPs packaged in the virus particles remain tightly associated during their transport towards the nucleus. (3) Upon nuclear import, the associated vRNPs of different segments disassemble in the nucleus (Results in Figure 3) and (4) different vRNAs replicate in different areas of the nucleus and no segment-specific accumulation of vRNAs is observed (Results in Figure 2&3). (5) The newly synthesized vRNPs are exported into the cytoplasm individually and different segments are not associated with each other shortly after their nuclear export (Results in Figure 2&3). (6) During the vRNP trafficking to the plasma membrane, the vRNPs are loaded onto recycling endosomes through the association with Rab11 [15], [16], [17]. The vRNPs of different segments then start to colocalize with each other and travel together towards the apical plasma membrane as a pre-formed complex of eight different vRNPs. (7) As the complex reaches the plasma membrane, the eight different vRNPs are then packaged as a whole into the budding virions.
Mentions: In this study, we have analyzed the disassembly and the subsequent assembly of influenza vRNPs segments in virus-infected cells using smFISH. We show that vRNPs of different segments remain associated after their release from the incoming virions and they travel as a package to the nuclear membrane. Newly synthesized vRNPs of different segments do not occupy the same space in the nucleus and they are likely exported individually into the cytoplasm because colocalization of the exported vRNPs of different segments is not observed during the early stages of infection. Different viral RNPs colocalize in the cytoplasm at later stages during infection (6–8 hpi in MDCK cells) and they are often found to be associated with Rab11-recycling endosomes. These results provide evidence that vRNPs belonging to different segments follow the same trafficking route and the selection for the correct combination of the eight vRNA segments likely takes place before the vRNPs reach the cell surface (Fig. 7).

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