Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells.

We show that a cellular nuclear protein, the SR splicing factor SF2/ASF, controls the level of production of an essential influenza virus protein, the M2 ion channel protein. The M2 mRNA that encodes the ion channel protein is produced by alternative splicing of another viral mRNA, M1 mRNA. The production of M2 mRNA is controlled in two ways. First, a distal (stronger) 5' splice site in M1 mRNA is blocked by the complex of viral polymerase proteins synthesized during infection, allowing the cellular splicing machinery to switch to the proximal (weaker) M2 5' splice site. Second, utilization of the weak M2 5' splice site requires its activation by the cellular SF2/ASF protein. This activation is mediated by the binding of the SF2/ASF protein to a purine-rich splicing enhancer sequence that is located in the 3' exon of M1 mRNA. We demonstrate that activation of the M2 5' splice site is controlled by the SF2/ASF protein in vivo during influenza virus infection. Utilizing four cell lines that differ in their levels of production of the SF2/ASF protein, we show that during virus infection of these cell lines both M2 mRNA and the M2 ion channel protein are produced in amounts that are proportional to the different expression levels of the SF2/ASF protein.     

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs.

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).     

Ion selectivity and activation of the M2 ion channel of influenza virus.

The influenza A virus-associated M2 ion channel is generally believed to function during uncoating of virions in infected cells. On endocytosis of a virion into the lumen of endosomes, the M2 ion channel is thought to cause acidification of the virion interior. In addition, the influenza virus M2 ion channel is thought to function in the exocytic pathway by equilibrating the pH gradient between the acidic lumen of the trans-Golgi network and the neutral cytoplasm. A necessary test of the proposed roles of the influenza virus M2 ion channel in the virus life cycle is to show directly that the M2 ion channel conducts protons. We have measured the ionic selectivity and activation of three subtypes (Udorn, Weybridge, and Rostock) of the M2 ion channel in oocytes of Xenopus laevis by measurement of 1) the intracellular pH (pHin) of voltage-clamped oocytes, 2) the current-voltage relationship in solutions of various pH and ionic composition, and 3) the flux of 86Rb. We took advantage of the low pHin achieved during incubation in low pH medium to study the effects of low pHin on M2 activation. Oocytes expressing each of the three subtypes of the M2 protein a) underwent a slow acidification when incubated in medium of low pH (acidification was blocked by the M2 ion channel inhibitor, amantadine); b) had current-voltage relationships that shifted to more positive values and had greater conductance when the pHout was lowered (this relationship was modified when Na- was replaced by NH4+ or Li+); c) had an amantadine-sensitive influx of Rb+. The effects on the current-voltage relationship of reduced pHin were opposed to the increased conductance found with reduced pHout. We interpret these results to indicate that the M2 ion channel is capable of conducting H+ and that other ions may also be conducted. Moreover, the channel conductance is reduced by decreased pHin. These findings are consistent with the proposed roles of the M2 protein in the life cycle of influenza A virus.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.     

A型流感病毒H5N1的M2离子通道稳定细胞株的建立

A型流感病毒H5N1的M2离子通道(H5M2)基因经优化后由人工合成,适合于哺乳动物细胞中表达.通过酶切克隆于pcDNA4质粒,并在HEK293细胞中建立稳定细胞株.Western blotting和免疫荧光证实H5M2在稳定细胞中只有在四环素诱导下才能表达,并经膜片钳证实在HEK293细胞中表达的H5M2具有H 通道活性,为M2离子通道功能的研究和M2离子通道阻断剂筛选方法的建立提供了参考.     

Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types.     

Modification of eukaryotic initiation factor 4F during infection by influenza virus.

Influenza virus infection of cells is accompanied by a striking shutoff of cellular protein synthesis, resulting in the exclusive translation of viral mRNAs. The mechanism for control of cellular protein synthesis by influenza virus is poorly understood, but several translation properties of influenza virus mRNAs which are potentially involved have been described. Influenza virus mRNAs possess the surprising ability to translate in the presence of inhibitory levels of inactive (phosphorylated) eukaryotic initiation factor 2 (eIF-2). In addition, influenza virus mRNAs were shown to be capable of translating in cells during the late phase of adenovirus infection but not in cells infected by poliovirus. Since both adenovirus and poliovirus facilitate virus-specific translation by impairing the activity of initiation factor eIF-4F (cap-binding protein complex) but through different mechanisms, we investigated the translation properties of influenza virus mRNAs in more detail. We show that influenza virus infection is associated with the significant dephosphorylation and inactivation of eIF-4E (cap-binding protein), a component of eIF-4F, and accordingly that influenza virus mRNAs possess a moderate ability to translate by using low levels of eIF-4F. We also confirm the ability of influenza virus mRNAs to translate in the presence of high levels of inactive (phosphorylated) eIF-2 but to a more limited extent than reported previously. We suggest a potential mechanism for the regulation of protein synthesis by influenza virus involving a decreased requirement for large pools of active eIF-4F and eIF-2.     

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA₃, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.     

The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells

The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.     

Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production

The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.     

Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation.

The interferon-regulated mouse Mx gene encodes the 72-kilodalton nuclear Mx protein that selectively inhibits influenza virus replication. Mice carrying Mx+ alleles synthesize Mx protein and resist influenza virus infection, whereas mice homozygous for Mx- alleles fail to synthesize Mx protein and, as a consequence, are influenza virus susceptible. Southern blot analysis allowed us to define the following three distinct Mx restriction fragment length polymorphism (RFLP) types among classical inbred strains: RFLP type 1 in the Mx+ strains A2G and SL/NiA, RFLP type 2 in BALB/c and 33 other Mx- strains, and RFLP type 3 in CBA/J and 2 other Mx- strains. cDNA clones of Mx mRNAs from BALB/c and CBA/J cells were isolated, and their sequences were compared with that of the wild-type Mx mRNA of strain A2G. Mx mRNA of BALB/c mice has 424 nucleotides absent from the coding region, resulting in a frame shift and premature termination of Mx protein. The missing sequences correspond exactly to Mx exons 9 through 11. These three exons, together with some flanking intron sequences, are deleted from the genomes of all Mx RFLP type 2 strains. The Mx- phenotype of the Mx RFLP type 3 strain CBA/J is due to a point mutation that converts the lysine codon in position 389 to a termination codon. Mx RFLP type 3 strains have an extra HindIII site which maps to an intron and thus probably does not affect the coding capacity of Mx mRNA. We further show that the Mx mRNA levels in interferon-treated BALB/c and CBA/J cells are about 15-fold lower than in similarly treated Mx+ cells. This is probably due to decreased metabolic stabilities of the mutant mRNAs.     

Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport.

The influenza A/fowl plague virus/Rostock/34 hemagglutinin (HA), which is cleaved intracellularly and has a high pH threshold (pH 5.9) for undergoing its conformational change to the low-pH form, was expressed from cDNA in CV-1 and HeLa T4 cells in the absence of other influenza virus proteins. It was found, by biochemical assays, that the majority of the HA molecules were in a form indistinguishable from the low-pH form of HA. The acidotropic agent, ammonium chloride, stabilized the accumulation of HA in its native form. Coexpression of HA and the homotypic influenza virus M2 protein, which has ion channel activity, stabilized the accumulation of HA in its pH neutral (native) form, and the M2 protein ion channel blocker, amantadine, prevented the rescue of HA in its native form. These data provide direct evidence that the influenza virus M2 protein ion channel activity can affect the status of the conformational form of cleaved HA during intracellular transport.