Summary auto-generated
This study investigated how influenza virus NS1 protein alters the nuclear organization of cellular RNA splicing components. Using immunofluorescence microscopy in infected MDCK cells, researchers observed that during infection, the normal speckled distribution pattern of the splicing factor SC35 became disorganized and punctate, while coiled bodies (nuclear structures enriched in splicing machinery) increased in number but decreased in size. Fibrillarin staining remained unchanged, indicating these changes were specific to splicing components rather than global nuclear disruption. Transfection experiments with plasmids expressing NS1 protein alone reproduced the alterations in SC35 and small nuclear ribonucleoprotein (snRNP) distribution without increasing coiled bodies, indicating NS1 protein directly causes these changes. The NS2 protein control showed no effect. These structural reorganizations correlate with previously reported NS1-mediated inhibition of mRNA splicing, suggesting NS1 protein directly interacts with components of the splicing machinery, possibly interfering with the second catalytic step of the splicing reaction.
Key findings
- Influenza virus NS1 protein alone causes redistribution of splicing factor SC35 from normal speckled pattern to diffuse punctate distribution
- NS1 protein alters nuclear localization of snRNPs, causing disappearance of characteristic pattern and diffuse nuclear staining
- NS1-mediated splicing inhibition correlates with altered intranuclear organization of splicing components, suggesting direct interaction with splicing machinery
- Increased coiled body numbers during infection result from general stimulation of gene expression rather than NS1-specific effects
- These structural changes are specific to splicing components; nucleolar proteins like fibrillarin show no alterations during infection
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Abstract
Intranuclear structure was studied in influenza virus-infected cells by immunofluorescence microscopy with antibodies specific for fibrillarin, the splicing factor SC35 and the autoantigen p80 coilin. In the course of the infection, an increase in the number of coiled bodies was observed, with a parallel decrease in their size. In addition, the normal speckled pattern of the SC35 factor was altered to generate a more punctate distribution. However, no alteration was observed in the fibrillarin staining pattern. Since an alteration in the splicing of both viral and cellular mRNAs upon expression of influenza virus NS1 protein has been reported previously, the possible effects of NS1 expression on intranuclear structure were assayed. The increase in the coiled body numbers was not specific for the expression of NS1 protein, but alterations in the nuclear location of small ribonucleoprotein particles, as determined by immunofluorescence with an anti-Sm serum or the SC35 splicing factor, were produced by the sole expression of NS1 protein. These results correlate with the previously reported inhibition of splicing induced by NS1 protein expression and suggest an interaction of this influenza virus protein with the cellular splicing machinery.