Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons (original) (raw)
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Transport of incoming influenza virus nucleocapsids into the nucleus
Trends in Cell Biology, 1992
Upon penetration of the influenza virus nucleocapsid into the host cell cytoplasm, the viral RNA and associated proteins are transported to the nucleus, where viral transcription and replication occur. By using quantitative confocal microscopy, we have found that over half of cell-associated nucleoprotein (NP) entered the nucleus with a half time of 10 min after penetration into CHO cells. Microinjection and immunoelectron microscopy experiments indicated that the NP entered the nucleus through the nuclear pore as part of an intact ribonucleoprotein (RNP) structure and that its transport was an active process. Transport of the incoming RNPs into the nucleus was not dependent on an intact microfilament, microtubule, or intermediate filament network. Subsequent to penetration, the matrix (MI) protein appeared to dissociate from the RNP structure and to enter the nucleus independently of the RNP. We found that 50% of penetrated Ml entered the nucleus with a half time of 25 min after penetration into CHO cells. Nuclear transport of Ml appeared to occur by passive diffusion. Entry of incoming Ml into the nucleus was not a prerequisite for infection.
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...
The role of nuclear import and export in influenza virus infection
Trends in Cell Biology, 1996
Many enveloped viruses, including influenza virus, herpes viruses, hepatitis B virus and RNA tumour viruses, replicate their genomes in the nucleus, but the virus particles themselves mature at the plasma membrane or at cytoplasmic membranes. Because of the spatial separation between genome replication and viral maturation, these viruses must not only get their genomes into the nucleus to initiate infection, but must also ensure that, after replication, the genome is transported to cytosolic budding sites. Each of these virus families use different strategies to solve the nuclear trafficking problem.
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...
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.
Virus Research, 2003
Most RNA viruses that lack a DNA phase replicate in the cytoplasm. However, several negative-stranded RNA viruses such as influenza, Thogoto, and Borna disease viruses replicate their RNAs in the nucleus, taking advantage of the host cell's nuclear machinery. A challenge faced by these viruses is the trafficking of viral components into and out of the nucleus through the nuclear membrane. The genomic RNAs of these viruses associate with proteins to form large complexes called viral ribonucleoproteins (vRNPs), which exceed the size limit for passive diffusion through the nuclear pore complex (NPC). To insure efficient transport across the nuclear membrane, these viruses use nuclear import and export signals exposed on the vRNPs. These signals recruit the cellular import and export complexes, which are responsible for the translocation of the vRNPs through the NPC. The ability to control the direction of vRNP trafficking throughout the viral life cycle is critical. Various mechanisms, ranging from simple posttranslational modification to complex, sequential masking-and-exposure of localization signals, are used to insure the proper movement of the vRNPs. #
A Classical Bipartite Nuclear Localization Signal on Thogoto and Influenza A Virus Nucleoproteins
We have previously shown that the nucleoprotein (NP) of Thogoto virus (THOV), a tick-borne member of the Orthomyxoviridae family, accumulates in the cell nucleus. Here we demonstrate that THOV NP contains a motif (KRxxxxxxxxxKTKK) at amino acid positions 179±193 that represents a classical bipartite nuclear localization signal (NLS). This sequence motif (named cNLS) was able to translocate a cytoplasmic 80-kDa reporter protein into the nucleus. Targeted mutations substituting lysines for alanines in the downstream cluster of the bipartite motif abolished the capacity of cNLS to mediate nuclear import. In contrast, identical mutations had no effect on nuclear localization when introduced into THOV NP, indicating that additional transport signals are present in NP. Amino-acid sequence comparisons revealed that THOV NP lacks the N-terminal nonconvential NLS (named here nNLS), which has been implicated in nuclear import of influenza A virus NP. Accordingly, THOV NP failed to interact in coprecipitation assays with the cellular NPI-1/3 transport factors of the karyopherin ␣ family. A highly conserved motif identified in THOV NP was the so-called nuclear accumulation sequence (NAS). Mutating NAS alone, or in combination with cNLS, had no gross effect on the intracellular distribution of the protein, indicating that a functional NAS is not required for nuclear accumulation of THOV NP in mammalian cells. We also studied nuclear transport of influenza A/PR/8/34 virus NP. Interestingly, we found a cNLS motif at amino acid positions 198±216 in addition to the previously described nonconventional nNLS. To further assess the functional role of cNLS, nNLS, and NAS, we analyzed single, double, and triple mutants of influenza A virus NP. When nNLS was destroyed, the protein stayed in the cytoplasm as expected. When NAS was disrupted in addition to nNLS, the double mutant accumulated in the nucleus, suggesting that cNLS was active. Indeed, when cNLS was also inactivated, the triple mutant protein localized again predominantly to the cytoplasm. These findings suggest that NP of orthomyxoviruses have two independent NLSs, namely cNLS and nNLS. They further suggest that NAS and NLSs may assume opposing roles in nucleocytoplasmic transport of NP.
Journal of Virology, 1995
The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated...
Journal of Virology, 2015
Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection.