Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons.

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 infection, it was also found to be a shuttling protein.     

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.     

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus.

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 by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.     

Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA.

The biosynthesis, nuclear transport, and formation of a complex among the influenza polymerase proteins were studied in influenza virus-infected MDBK cells by using monospecific antisera. To obtain these monospecific antisera, portions of cloned cDNAs encoding the individual polymerase proteins (PB1, PB2, or PA) of A/WSN/33 influenza virus were expressed as fusion proteins in Escherichia coli, and the purified fusion proteins were injected into rabbits. Studies using indirect immunofluorescence showed that early in the infectious cycle (4 h postinfection) of influenza virus, PB1 and PB2 are present mainly in the nucleus, whereas PA is predominantly present in the cytoplasm of the virus-infected cells. Later, at 6 to 8 h postinfection, all three polymerase proteins are apparent both in the cytoplasm as well as the nucleus. Radiolabeling and immunoprecipitation analyses showed that the three polymerase proteins remain physically associated as a complex in either the presence or the absence of ribonucleoproteins. In the cytoplasm, the majority of the polymerase proteins remain unassociated, whereas in the nucleus they are present as a complex of three polymerase proteins. To determine whether a polymerase protein is transported into the nucleus individually, PB1 was expressed from the cloned cDNA by using the simian virus 40 late promoter expression vector. PB1 alone, in the absence of the other polymerase proteins or the nucleoprotein, accumulates in the nucleus. This suggests that the formation of a complex with other viral protein(s) is not required for either nuclear transport or nuclear accumulation of PB1 protein and that the PB1 protein may contain an intrinsic signal(s) for nuclear transport.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Analysis of Nuclear Accumulation of Influenza NP Antigen in von Magnus Virus-Infected Cells

When 1–5C-4 cells were infected with von Magnus virus derived from influenza A/RI/5⁺ virus by four successive undiluted passages in chick embryos, virus-specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus-infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus-infected cells NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus-infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble form and the amount of cytoplasmic RNP was considerably reduced. After short-pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus-infected cells is not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.     

Intracellular development of membrane protein of influenza virus.

The intracellular development of membrane protein (MP) of influenza A virus was investigated by immunofluorescent staining. Monospecific antiserum was prepared by immunizing rabbits with MP eluted from SDS-polyacrylamide gels of SDS-disrupted NWS virions. In the productive infection in clone 1-5C-4 cells, MP antigen was first detected over the whole cell at 4 hr after infection, concomitantly with the appearance of hemagglutinin (HA) antigen in the cytoplasm, and bright nuclear fluorescence was then observed. Nucleoprotein (NP) antigen was detected in the nucleus prior to the appearance of fluorescence of MP antigen and thereafter the cytoplasmic fluorescence developed. Late in infection, all of these three antigens were observed predominantly in the cytoplasm with stronger fluorescence at the cell surface. Essentially similar findings were obtained in the abortive infections in L cells and BHK cells. The above results suggest that the membrane protein of influenza A virus is present in the nucleus as well as in the cytoplasm of infected cells.     

The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.     

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.     

A Rab11- and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNA

The viral RNA (vRNA) genome of influenza A virus is replicated in the nucleus, exported to the cytoplasm as ribonucleoproteins (RNPs), and trafficked to the plasma membrane through uncertain means. Using fluorescent in situ hybridization to detect vRNA as well as the live cell imaging of fluorescently labeled RNPs, we show that an early event in vRNA cytoplasmic trafficking involves accumulation near the microtubule organizing center in multiple cell types and viral strains. Here, RNPs colocalized with Rab11, a pericentriolar recycling endosome marker. Cytoplasmic RNP localization was perturbed by inhibitors of vesicular trafficking, microtubules, or the short interfering RNA-mediated depletion of Rab11. Green fluorescent protein (GFP)-tagged RNPs in living cells demonstrated rapid, bidirectional, and saltatory movement, which is characteristic of microtubule-based transport, and also cotrafficked with fluorescent Rab11. Coprecipitation experiments showed an interaction between RNPs and the GTP-bound form of Rab11, potentially mediated via the PB2 subunit of the polymerase. We propose that influenza virus RNPs are routed from the nucleus to the pericentriolar recycling endosome (RE), where they access a Rab11-dependent vesicular transport pathway to the cell periphery.     

Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins.

We investigated the properties of ts51, an influenza virus (A/WSN/33) temperature-sensitive RNA segment 7 mutant. Nucleotide sequence analysis revealed that ts51 possesses a single nucleotide mutation, T-261----C, in RNA segment 7, resulting in a single amino acid change. Phenylalanine (position 79) in the wild-type M1 protein was substituted by serine in ts51. This mutation was phenotypically characterized by dramatic nuclear accumulation of the M1 protein and interfered with some steps at the late stage of virus replication, possibly affecting the assembly and/or budding of viral particles. However, although M1 protein was retained within the nucleus, export of the newly synthesized viral ribonucleoprotein containing the minus-strand RNA into the cytoplasm was essentially the same at both permissive and nonpermissive temperatures. The roles of M1 in the export of viral ribonucleoproteins from the nucleus into the cytoplasm and in the virus particle assembly process are discussed.     

Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import

Because influenza virus replicates in the nucleus and buds from the plasma membrane, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear membrane. Export from the nucleus to the cytoplasm was found to depend on the viral matrix protein (M1). M1 associated with newly assembled viral RNPs (vRNPs) in the nucleus and escorted them to the cytoplasm through the nuclear pores. In contrast, during entry of the virus into a new host cell, M1 protein dissociated from the RNPs, allowing them to enter the nucleus. Amantadine, an antiviral agent that induces an early block in influenza A infection, was found to block the dissociation event and thereby to prevent import of incoming RNPs into the nucleus. Together, these results showed that M1 modulates the directionality of vRNP transport into and out of the nucleus.     

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.     

The NPI-1/NPI-3 (karyopherin alpha) binding site on the influenza a virus nucleoprotein NP is a nonconventional nuclear localization signal.

Two cellular proteins, NPI-1 and NPI-3, were previously identified through their interaction with the influenza virus nucleoprotein (NP) by using the yeast two-hybrid system. These proteins were then shown to act as general transport factors (karyopherin alpha) and nuclear pore-docking proteins to facilitate the transport of the NP and of viral RNA into the nucleus. The yeast two-hybrid assay has now been used to identify the specific domains on the NP that bind to the NPI proteins. Mutational analysis including alanine scanning identified the motifs SxGTKRSYxxM and TKRSxxxM, which are required for binding to NPI-1 and NPI-3, respectively. These sequences were shown to possess nuclear localization signal (NLS) activity following expression of fusion proteins in HeLa cells. These sequences represent a novel nonconventional NLS motif. Another NLS activity not mediated by the NPI binding sites is associated with noncontiguous sequences in the NP.     

A nuclear export signal in the matrix protein of Influenza A virus is required for efficient virus replication

The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication.