Mutations in matrix protein 1 and nucleoprotein caused human-specific defects in nuclear exportation and viral assembly of an avian influenza H7N1 virus (original) (raw)
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Role of the Influenza Virus M1 Protein in Nuclear Export of Viral Ribonucleoproteins
Journal of Virology, 2000
The protein kinase inhibitor H7 blocks influenza virus replication, inhibits production of the matrix protein (M1), and leads to a retention of the viral ribonucleoproteins (vRNPs) in the nucleus at late times of infection (K. Martin and A. Helenius, Cell 67:117–130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional f...
Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons
Journal of Virology, 1996
The influenza virus nucleoprotein (NP), matrix protein (M1), and ribonucleoproteins (vRNPs) undergo regulated nuclear import and export during infection. Their trafficking was analyzed by using interspecies heterokaryons containing nuclei from infected and uninfected cells. Under normal conditions, it was demonstrated that the vRNPs which were assembled in the nucleus and transported to the cytosol were prevented from reimport into the nucleus. To be import competent, they must first assemble into virions and enter by the endosomal entry pathway. In influenza virus mutant ts51, in which M1 is defective, direct reimport took place but was inhibited by heterologous expression of wild-type M1. These data confirm M1's role as the inhibitor of premature nuclear import and as the main regulator of nuclear transport of vRNPs. In addition to this vRNP shuttling, M1 also shuttled between the nucleus and the cytoplasm in ts51-infected cells. When NP was expressed in the absence of virus i...
The Journal of general virology, 2000
A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the tra...
Virology Journal
Background: The influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection. N-domain and C-domain are connected by a loop region, which consists of conserved RQMV motif. Methods: The function of the highly conserve RQMV motif in the influenza virus life cycle was investigated by sitedirected mutagenesis and by rescuing mutant viruses by reverse genetics. Co-localization of M1 with nucleoprotein (NP), clustered mitochondria homolog protein (CLUH), chromosome region maintenance 1 protein (CRM1), or plasma membrane were studied by confocal microscopy. Results: Mutant viruses containing an alanine substitution of R163, Q164 and V166 result in the production of the virus indistinguishable from the wild type phenotype. Single M165A substitution was lethal for rescuing infection virus and had a striking effect on the distribution of M1 and NP proteins. We have observed statistically significant reduction in distribution of both M165A (p‹0,05) and NP (p‹0,001) proteins to the nucleus in the cells transfected with the reverse-genetic system with mutated M1. M165A protein was co-localized with CLUH protein in the cytoplasm and around the nucleus but transport of M165-CLUH complex through the nuclear membrane was restricted. Conclusions: Our finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.
Journal of virology, 1999
The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the o...
Virus Research, 1985
The influenza virus nucleoprotejn gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circula~zed by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA : dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known. mRNA cloning, gene expression, DNA transfect~on, specific priming
Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export
Virology Journal, 2014
Background: Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Methods: Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Results: Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. Conclusions: These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we conclude that in both A549 and 293 T cells, PA, PB1, and PB2 mRNA nuclear export is Nxf1 and Crm1 independent. Our data support the hypothesis that PA, PB1, and PB2 mRNAs, encoding the influenza RdRP, utilize atypical mRNA nuclear export.
Functional expression of influenza A viral nucleoprotein in cells transformed with cloned DNA
Virology, 1986
Simian cells permissive for influenza A virus infection were stably transformed with a full-length cloned influenza A nucleoprotein gene under the control of an inducible metallothionein promoter and linked to a dihydrofolate reductase gene to facilitate cell selection. Transformed cells synthesized a virus-specific nucleoprotein which was indistinguishable from the nucleoprotein synthesized in virus-infected cells with respect to molecular weight and intracellular localization. It was estimated that transformed cells produced only 1% of the amount of nucleoprotein synthesized in simian cells infected with influenza A virus. Nontheless, when transformed cells were infected with influenza virus mutants which synthesized temperature-sensitive nucleoprotein, protein expressed by the cloned gene was able to complement the synthesis of plus-strand and minus-strand viral RNA for one mutant and only plus-strand synthesis for another mutant. This indicated that the influenza A nucleoprotein expressed in the transformed cells exhibited functional activity.
Characterisation of an avian influenza virus nucleoprotein expressed inE. coli and in insect cells
Archives of Virology, 1990
The nucleoprotein (NP) gene from influenza virus A/Shearwater/ Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. col#derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive-and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.
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...