Many different viruses target the nucleolus to disrupt host-cell functions and to recruit cellular proteins to aid in virus replication. As the primary site of the replication of positive-strand RNA viruses and most negative-strand RNA viruses is the cytoplasm, the reason for RNA viruses to target nuclear structures is not immediately obvious. However, among RNA viruses, influenza virus is an exception, since all the essential viral replication events take place in the nucleus.
We have previously shown that the NS1 protein of the H3N2 subtype influenza A virus contains a C-terminal NLS2/NoLS that efficiently targets the protein into the nucleoli in virus-infected cells
. Murayama and co-workers identified the nucleolar protein, nucleolin, as a novel NS1-binding protein
. They also suggested that the N-terminal part of the NS1 protein was responsible for this binding, as judged by a GST pull-down assay with the GST-fused functional domains of NS1. By laser confocal microscopy, they observed that NS1 colocalized with nucleolin most clearly in the nucleoli, indicating that NS1 was interacting with nucleolin during the infection
In the present study, instead, we show that the NS1 protein of the human H3N2 subtype influenza A virus can interact, primarily via its C-terminal NoLS, with the main nucleolar proteins, nucleolin, B23 and fibrillarin in vitro. Using GFP-NS1 fusion proteins, we observed that the N-terminal part of both the H3N2 and H1N1 subtype influenza NS1 protein, containing a functional NLS1, targeted the chimeric proteins into the cell nucleus but not to the nucleolus (Figure
2B and C). However, only the C-terminus of the NS1 protein of the H3N2 subtype A influenza virus, containing a functional NLS2/NoLS, targeted the chimeric protein into the cell nucleus and nucleoli (Figure
2B and C). Mutations in the NLS2/NoLS abolished both the nuclear and nucleolar targeting (Figure
2B). A chimeric GFP-NS1 A/Brevig Mission/1/18(203–230) protein was only faintly targeted into the cell nucleus and nucleolus (Figure
2C). These results are in agreement with our previous results showing that all types of influenza A virus NS1 proteins have an N-terminal NLS1, while H3N2 subtype viruses also have an additional NLS, a C-terminal NLS2, which also functions as a NoLS
As mentioned above, Murayama and co-workers showed that the N-terminal RNA-binding domain of the NS1 protein was responsible for the binding to nucleolin
. Using GST-NS1 A/Udorn/72(1–73) fusion protein construct and A549 cell extracts, we were also able to see a weak binding of nucleolin, B23 and fibrillarin to the N-terminal part of a chimeric NS1 protein (Figure
3). The binding was abolished by R38A and K41A substitution mutations to the RNA-binding domain of the NS1 protein (Figure
3B). The same amino acids are also critical for the importin α-binding and nuclear translocation of NS1 protein via the nuclear transport machinery
. Instead, the C-terminal part of the NS1 protein in the chimeric construct GST-NS1/A/Udorn/72(203–237) bound nucleolin, B23 and fibrillarin as well as the wt NS1 protein did (Figure
3B). This binding was totally abolished by K219A,R220A + R231A,R232A substitution mutations (Figure
3B), indicating the NLS2/NoLS to be primarily responsible for the binding of NS1 to nucleolar proteins.
The signals that govern the nucleolar localization and retention of different proteins are not well defined. The motifs involved are usually rich in arginine and lysine residues, however, there is no obvious consensus sequence or structure. Proteins that localize in the nucleolus can also have nuclear import and export motifs. It is unclear whether nucleolar proteins are specifically localized, targeted, accumulated or just retained in the nucleolus and consequently the sequences associated with the nucleolar localization have thus been designated as nucleolar targeting signals and/or nucleolar retention signals. In many cases, proteins that localize in the cytoplasm and in the nucleus and/or nucleoli contain multiple signals that determine their subcellular localization. This highlights the difficulty in identifying truly functional NoLSs. Moreover, proteins that localize in the nucleoli must first be imported into the nucleus and therefore they likely possess both NLS and NoLS whose sequences may overlap (Reviewed in
 and present data clearly shows that the nucleolar retention of the H3N2 subtype influenza A virus NS1 protein is mediated primarily by its C-terminal NLS2/NoLS. However, the N-terminal NLS1 may also mediate weak nuclear retention of NS1 protein, since the H1N1pdm NS1 protein was weakly localized into the nucleoli, even if it lacked the C-terminal NLS2/NoLS due to a stop codon at position 220 (Figure
1). There may also be viral species-specific differences, since the 230-amino-acid-long NS1 of the avian A/mallard/Netherlands/12/00 virus completely failed to target into the nucleoli (Figure
Nucleoli are compact structures, and thus it was quite expected that all three major nucleolar proteins co-precipitated with the H3N2 virus NS1 protein in GST pull-down experiments. However, in vitro-translated nucleolar proteins showed that nucleolin and fibrillarin specifically bound to the C-terminal part of the NS1 protein in a manner similar to that seen with A549 cell extracts (compare Figures
4). Instead, in vitro-translated B23 did not bind to the NS1 protein suggesting that this protein has no intrinsic binding ability to NS1. However, it coprecipitated with other nucleolar proteins in GST pull-down experiments (Figure
4). It is also possible that in vitro-translated B23 is misfolded or requires additional posttranslational modifications, such as phosphorylation for the NS1 interaction to take place.
Laser confocal microscopy showed a clear colocalization of NS1 with nucleolin, B23 and fibrillarin, indicating that NS1 was interacting with these proteins during the virus infection (Figure
5A). The HIV-1 Rev protein has previously been shown to localize into the nucleolus (reviewed in
). Like many viral proteins that target into the nucleolus, Rev has also been shown to contain a functional NoLS
. In the nucleolus, HIV-1 Rev protein binds to nucleolar B23
 and localizes to the combined regions of the dense fibrillar component and the granular component. To further identify the interaction partners of NS1 in the nucleolus, we analyzed nucleolar co-localization of the GFP-NS1 A/Udorn/72/(203–237) and HIV-1 Rev proteins. GFP-NS1 A/Udorn/72/(203–237) and HIV-1 Rev proteins colocalized in the nucleolus, and high expression level of the HIV-1 Rev protein totally displaced the chimeric GFP-NS1(203–237) protein from the nucleolus. This may indicate that these proteins are binding to similar structures within the nucleolus or that all nucleolar proteins have a very tight interaction with each others.
Further analysis of the role of the nuclear retention of the NS1 protein during influenza virus infection is needed. The fact that viral proteins contain NoLSs is an indication that viruses have evolved specific nucleolar functions. There is emerging evidence that the disruption of the nuclear or nucleolar trafficking by some positive-stranded RNA virus proteins may have a role in viral pathogenesis.