Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes (original) (raw)
Related papers
Virology journal, 2014
The influenza A virus NS1 protein is a virulence factor and an antagonist of host cell innate immune responses. During virus infection NS1 protein has several functions both in the nucleus and in the cytoplasm and its intracellular localization is regulated by one or two nuclear localization signals (NLS) and a nuclear export signal (NES). In order to investigate the role of NS1 NES in intracellular localization, virus life cycle and host interferon responses, we generated recombinant A/Udorn/72 viruses harboring point mutations in the NES sequence. NS1 NES was found to be inactivated by several of the mutations resulting in nuclear retention of NS1 at late stages of infection confirming that this sequence is a bona fide functional NES. Some of the mutant viruses showed reduced growth properties in cell culture, inability to antagonize host cell interferon production and increased p-IRF3 levels, but no clear correlation between these phenotypes and NS1 localization could be made. Im...
Journal of General Virology, 2010
During influenza A virus infection, the NS1 protein is engaged in different functions in different intracellular compartments. In this study, we showed that the NS1 of A/PR/8/34 localized in different positions from that of A/Sydney/5/97 when transiently expressed in Madin-Darby canine kidney cells. Residue 221 of NS1 was identified to be a new key residue involved in the Cterminal nuclear localization signal (NLS) and nucleolar localization signal (NoLS) of NS1 from A/ Sydney/5/97. Analysis of chimeric NS1 and further mutants showed that residues responsible for the binding between NS1 and the cleavage and polyadenylation specificity factor (CPSF) are correlated with the intracellular localization of transiently expressed NS1 proteins. Fluorescence loss in photobleaching imaging revealed that the NS1 protein with both functional NLSs and nuclear export signal (NES) was able to shuttle between the nucleus and cytoplasm. Drug inhibition experiments and fluorescence resonance energy transfer analysis suggested that NS1 was exported out of the cell nuclei via a Crm1-independent pathway. Moreover, it is likely that another cytoplasmic localization-related sequence exists in the NS1 protein other than the leucine-rich NES. These findings provide new insights into the mechanism of intracellular localization and trafficking of influenza A virus NS1 protein, which is important for understanding its function.
Virology Journal, 2012
Background: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions.
Expression and characterisation of the influenza A virus non-structural protein NS 1 in ye
Arch Virol, 1994
The influenza A virus non-structural protein NS1 was produced using a copper-inducible expression system in the yeast Saccharomyces cerevisiae. The protein produced had a molecular weight of 26 kDa by SDS-PAGE and was reactive with anti-NSl antisera. The recombinant NS1 protein was targetted to the nucleolus/nuclear envelope fraction of the yeast cell nucleus, showing that its localisation signals remain functional in yeast. In addition, immune-electron microscopy detected cytoplasmic inclusions reminiscent of those seen in cells infected with some influenza strains. The NS~ protein was shown to be capable of in vivo self-interaction which probably forms the basis of its propensity to form inclusions. Expression of the protein was found to be toxic to yeast cells expressing it, supporting a role for the protein in the shutdown of influenza virus-infected cells. Deletion mapping of NS~ pointed to 2 regions of the molecule being important for this toxicity: a basic C-terminal stretch which has been shown to act as a nuclear localisation signal, and an N-terminal region implicated in RNA binding.
PLoS ONE, 2011
The 2009 pandemic H1N1 influenza virus encodes an NS1 protein with 11 amino acids (aa) truncation at the C-terminus. The C-terminal tail of influenza virus NS1 protein constitutes a nucleolar localization signal (NoLS) and is the binding domain of the cellular pre-mRNA processing protein, poly(A)-binding protein II (PABII). Here, our studies showed that the C-terminaltruncated NS1 of the 2009 pandemic virus was inefficient at blocking host gene expression, extension of the truncated NS1 to its full length increased the inhibition of host gene expression. Mechanistically, this increased inhibition of host gene expression by the full-length NS1 was not associated with nucleolar localization, but was due to the restoration of NS1's binding capacity to PABII. Furthermore, in vitro and in vivo characterization of two recombinant viruses encoding either the C-terminal 11-aa truncated or full-length NS1 of the 2009 pandemic virus showed that the C-terminal 11-aa truncation in NS1 did not significantly alter virus replication, but increased virus pathogenicity in mice.
Interaction of influenza A virus NS2/NEP protein with the amino-terminal part of Nup214
TURKISH JOURNAL OF BIOLOGY, 2020
Influenza A viruses have a single-stranded RNA genome consisting of 8 segments. Each RNA segment associates with the nucleoprotein (NP) and viral RNA polymerase to and from a viral ribonucleoprotein (vRNP) particle. The viral mRNA synthesis is dependent on a capped primer derived from nascent host RNA transcripts. For these processes to take place, vRNPs must pass through the cell nuclear pore complex (NPC) to the nucleus. The influenza A virus NS2 protein, also called the nuclear export protein (NES), has an important role in the nucleocytoplasmic transport of vRNPs. This protein interacts with the host cellular nucleoporins during the nuclear export of vRNPs. In this study, the human nucleoporin 214 (Nup214) was identified as an NS2-binding protein by using a yeast two-hybrid assay. The interaction between NS2 and human Nup214 was confirmed in both yeast and mammalian cells. It has been shown that the NS2 protein interacts with the amino terminal FG domain of the Nup214 protein. The influenza viral replication was suppressed in knockdown cells for the Nup214 protein. It was concluded that the FG domains of nucleoporins have an important role in the interaction of the influenza NS2 protein with host NPC for vRNA export.
Expression and analysis of the NS2 protein of influenza A virus
Archives of Virology, 1995
Influenza N S 2 protein was expressed in Saccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS 2 antiserum and was locatised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS 2 and the M 2 protein of the virus, involving the C-terminal 163 residues of M v A filter-binding assay localised the M~ binding region to the Cterminal 70 amino acids of NS r
Expression and characterisation of the influenza A virus non-structural protein NS1 in yeast
Archives of Virology, 1994
The influenza A virus non-structural protein NS1 was produced using a copper-inducible expression system in the yeast Saccharomyces cerevisiae. The protein produced had a molecular weight of 26 kDa by SDS-PAGE and was reactive with anti-NSl antisera. The recombinant NS1 protein was targetted to the nucleolus/nuclear envelope fraction of the yeast cell nucleus, showing that its localisation signals remain functional in yeast. In addition, immune-electron microscopy detected cytoplasmic inclusions reminiscent of those seen in cells infected with some influenza strains. The NS~ protein was shown to be capable of in vivo self-interaction which probably forms the basis of its propensity to form inclusions. Expression of the protein was found to be toxic to yeast cells expressing it, supporting a role for the protein in the shutdown of influenza virus-infected cells. Deletion mapping of NS~ pointed to 2 regions of the molecule being important for this toxicity: a basic C-terminal stretch which has been shown to act as a nuclear localisation signal, and an N-terminal region implicated in RNA binding.
Traffic (Copenhagen, Denmark), 2005
Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are nec...