A statistical framework for quantifying the nuclear export rate of influenza viral mRNAs
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Curated by eLife
eLife assessment
This important study combines virology experiments and mathematical modeling to determine the nuclear export rate of each of the eight RNA segments of the influenza A virus, leading to the proposal that a specific retention of mRNA within the nucleus delays the expression of antigenic viral proteins. The proposed model for explaining the differential rate of export is compelling, going beyond the state of the art, but the experimental setup is incomplete and would benefit from additional approaches. The insight so far is interesting, but because in the end it is left as an observation, the overall advance remains limited.
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Abstract
Influenza A virus acutely transcribes viral mRNAs from the eight segmented viral genome when it infects. The kinetics of viral transcription, nuclear export of viral transcripts, and their potential variation between the eight segments are poorly characterised. Here we introduce a statistical framework for estimating the nuclear export rate of each segment from a snapshot of mRNA in situ localisation at a single time point, exploiting the cell-to-cell variation observed by an imaging-based in situ transcriptome assay. A mathematical modelling indicated that the relationship between the nuclear ratio and the total count of mRNAs in single cells is dictated by a proxy for the nuclear export rate. Using this model, we showed that the two influenza viral antigens hemagglutinin and neuraminidase were the slowest segments in the nuclear export, suggesting that influenza A virus uses the nuclear retention of viral transcripts to delay the expression of antigenic molecules. Our framework presented in this study can be widely used for investigating the nuclear retention of nascent transcripts produced in a transcription burst.
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Author Response:
We thank the reviewers and eLife editorial team for their valuable assessment. While additional experiments could further strengthen the theoretical framework proposed in this study, we believe that we have successfully established the delayed nuclear export of hemagglutinin and neuraminidase mRNAs by quantifying the FISH observation with the mathematical model. We agree that this finding raises a further important question to be addressed regarding the molecular mechanism underlying the prolonged nuclear retention of these segments. Our ongoing investigation is focusing on identifying potential cis-elements that contribute to the delay of these segments.
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eLife assessment
This important study combines virology experiments and mathematical modeling to determine the nuclear export rate of each of the eight RNA segments of the influenza A virus, leading to the proposal that a specific retention of mRNA within the nucleus delays the expression of antigenic viral proteins. The proposed model for explaining the differential rate of export is compelling, going beyond the state of the art, but the experimental setup is incomplete and would benefit from additional approaches. The insight so far is interesting, but because in the end it is left as an observation, the overall advance remains limited.
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Reviewer #1 (Public Review):
The authors have approached the study of the mechanism by which the two more antigenic proteins of the influenza A virus, hemagglutinin (HA) and neuraminidase (NA), are expressed later during the infection. For this aim, they set an experimental approach consisting of a 2-hour-long infection at a multiplicity of infection of 2 with the viral strain WSN. They used cells from the lung carcinoma cell line A549. They used the FISH technique to detect the mRNAs in situ and developed an imaging-based assay for mathematically modeling and estimating the nuclear export rate of each of the eight viral segments. They propose that the delay in the expression of HA and NA is based on the retention of their mRNA within the nucleus.
The main strength of this work is the fact that the authors have studied a …
Reviewer #1 (Public Review):
The authors have approached the study of the mechanism by which the two more antigenic proteins of the influenza A virus, hemagglutinin (HA) and neuraminidase (NA), are expressed later during the infection. For this aim, they set an experimental approach consisting of a 2-hour-long infection at a multiplicity of infection of 2 with the viral strain WSN. They used cells from the lung carcinoma cell line A549. They used the FISH technique to detect the mRNAs in situ and developed an imaging-based assay for mathematically modeling and estimating the nuclear export rate of each of the eight viral segments. They propose that the delay in the expression of HA and NA is based on the retention of their mRNA within the nucleus.
The main strength of this work is the fact that the authors have studied a long-unaddressed mechanism in influenza A virus infectious cycle, as is the late expression of HA and NA, by creating a work flow including mRNA detection (FISH) plus mathematical calculations to arrive at a model, which additionally could be useful for general biological processes where transcription occurs in a burst-like manner. The weakness of this work in its present state is that in order to "quantify" the export rate of the transcripts, several assumptions regarding the viral infection are made without empirical data. It would greatly improve if more precise experiments could be performed and/or include demonstration of the assumptions made (i.e., synchronized infections, empirically demonstrating that cRNA production does not occur within the first 2 hours of infection, and/or separate transcription and replication, inhibiting RNA degradation during viral infection).
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Reviewer #2 (Public Review):
In this study the authors developed a framework to investigate the export rates of Influenza viral RNAs translocating from the nucleus to the cytoplasm. This model suggests that the influenza virus may control gene expression at the RNA export level, namely, the retention of certain transcripts in the nucleus for longer times, allows the generation of other viral encoded proteins that are exported regularly, and only later on do certain mRNAs get exported. These encode proteins that alert the cell to the presence of viral molecules, hence keeping their emergence to very end, might help the virus to avoid detection as late as possible in the infection cycle.
The study is of limited scope. The notion that some mRNAs are retained in the nucleus after transcription is concluded early on from the FISH data. The …
Reviewer #2 (Public Review):
In this study the authors developed a framework to investigate the export rates of Influenza viral RNAs translocating from the nucleus to the cytoplasm. This model suggests that the influenza virus may control gene expression at the RNA export level, namely, the retention of certain transcripts in the nucleus for longer times, allows the generation of other viral encoded proteins that are exported regularly, and only later on do certain mRNAs get exported. These encode proteins that alert the cell to the presence of viral molecules, hence keeping their emergence to very end, might help the virus to avoid detection as late as possible in the infection cycle.
The study is of limited scope. The notion that some mRNAs are retained in the nucleus after transcription is concluded early on from the FISH data. The model does not contribute much to the understanding and is mostly confirming the FISH data. The export rate is an ambiguous number and this part is not elaborated upon. One is left with more questions since no mechanistic knowledge emerges, and no additional experimentation is attempted to try drive to a deeper understanding.
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