Nuclear Export Of Viral Ribonucleoproteins Research Articles

4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1.

Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics.

Brevilin A, a Sesquiterpene Lactone, Inhibits the Replication of Influenza A Virus In Vitro and In Vivo.

With the emergence of drug-resistant strains of influenza A viruses (IAV), new antivirals are needed to supplement the existing counter measures against IAV infection. We have previously shown that brevilin A, a sesquiterpene lactone isolated from C. minima, suppresses the infection of influenza A/PR/8/34 (H1N1) in vitro. Here, we further investigate the antiviral activity and mode of action of brevilin A against different IAV subtypes. Brevilin A inhibited the replication of influenza A H1N1, H3N2, and H9N2 viruses in vitro. The suppression effect of brevilin A was observed as early as 4–8 hours post infection (hpi). Furthermore, we determined that brevilin A inhibited viral replication in three aspects, including viral RNA (vRNA) synthesis, expression of viral mRNA, and protein encoded from the M and NS segments, and nuclear export of viral ribonucleoproteins (vRNPs). The anti-IAV activity of brevilin A was further confirmed in mice. A delayed time-to-death with 50% surviving up to 14 days post infection was obtained with brevilin A (at a dose of 25 mg/kg) treated animals compared to the control cohorts. Together, these results are encouraging for the exploration of sesquiterpene lactones with similar structure to brevilin A as potential anti-influenza therapies.

In vitro anti-influenza virus activities of a new lignan glycoside from the latex of Calotropis gigantea.

A new lignan glycoside, (+)-pinoresinol 4-O-[6″-O-vanilloyl]-β-d-glucopyranoside (1) and two known phenolic compounds, 6′-O-vanilloyltachioside (2) and 6′-O-vanilloylisotachioside (3) were isolated from the latex of Calotropis gigantea (Asclepiadaceae). The structure of the new compound was elucidated by using spectroscopic and chemical methods. Three isolates (1–3) and one authentic compound, (+)-pinoresinol 4-O-β-d-glucopyranoside, were screened for A/PR/8/34 (H1N1) inhibitory activity by cytopathic effect (CPE) inhibition assay on MDCK cells. Compound 1 showed inhibitory activity against A/PR/8/34 (H1N1). In sharp contrast, the other three compounds (2, 3 and (+)-pinoresinol 4-O-β-d-glucopyranoside) did not show such activity. An analysis of structure-activity relationship between 1 and (+)-pinoresinol 4-O-β-d-glucopyranoside revealed that the presence of a vanilloyl group in the sugar moiety of 1 is crucial for its anti-influenza virus activity. Compound 1 was further evaluated for in vitro inhibitory activities against a panel of human and avian influenza viruses by CPE inhibition assay. It showed inhibitory effect against human influenza viruses in both subtypes A and B (IC50 values around 13.4–39.8 µM with SI values of 3.7–11.4), while had no effect on avian influenza viruses. Its antiviral activity against human influenza viruses subtype A was further confirmed by plaque reduction assay. The time course assay indicated that 1 exerts its antiviral activity at the early stage of viral replication. A mechanistic study showed that 1 efficiently inhibited influenza virus-induced activation of NF-κB pathway in a dose-dependent manner, but had no effect on virus-induced activation of Raf/MEK/ERK pathway. Further studies demonstrated that nuclear translocation of transcription factor NF-κB induced by influenza virus was significantly blocked by 1, meanwhile, nuclear export of viral ribonucleoproteins was also effectively inhibited. These findings suggest that this new lignan glycoside from Calotropis gigantea, may have therapeutic potential in influenza virus infection through inhibition of NF-κB pathway and viral ribonucleoproteins nuclear export.

Function of glycochains in virus infection

Influenza A virus (IAV) has two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA binds to sialic acids at the terminals of glycochains on the host cell surface as virus receptors. NA shows sialidase activity, which cleaves sialic acids from the terminals of glycochains. A new subtype (antigenicities of HA and NA) of IAV for humans has pandemic potential. We investigated the functions of HA and NA in IAV replication and pandemic potential in terms of glycoscience. We found that the sialidase activity of pandemic IAV had low pH stability, which enhanced IAV replication. It is thought that the low pH stability contributed to the pandemics in 1968 and 2009. HA also binds to sulfatide not containing sialic acid, and we found that sulfatide enhanced IAV replication. Binding of HA to sulfatide on the host cell surface enhanced progeny IAV formation in infected cells through the induction of the nuclear export of viral ribonucleoproteins by apoptosis. Sialic acid species are divided into N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The HAs of some human IAVs bind not only to Neu5Ac but also to Neu5Gc, which may facilitate the occurrence of a human IAV-based pandemic by genetic reassortment among IAV genomes in pig tracheas expressing Neu5Gc. We identified the amino acid residues of human IAV HA responsible for Neu5Gc binding and developed new techniques for the sensitive detection of IAV receptor specificities and infected cells. Our "glycovirology" research will provide new insights into the mechanisms of IAV replication and pandemic potential.

Influenza A virus attenuation by codon deoptimization of the NS gene for vaccine development.

Influenza viral infection represents a serious public health problem that causes contagious respiratory disease, which is most effectively prevented through vaccination to reduce transmission and future infection. The nonstructural (NS) gene of influenza A virus encodes an mRNA transcript that is alternatively spliced to express two viral proteins, the nonstructural protein 1 (NS1) and the nuclear export protein (NEP). The importance of the NS gene of influenza A virus for viral replication and virulence has been well described and represents an attractive target to generate live attenuated influenza viruses with vaccine potential. Considering that most amino acids can be synthesized from several synonymous codons, this study employed the use of misrepresented mammalian codons (codon deoptimization) for the de novo synthesis of a viral NS RNA segment based on influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus. We generated three different recombinant influenza PR8 viruses containing codon-deoptimized synonymous mutations in coding regions comprising the entire NS gene or the mRNA corresponding to the individual viral protein NS1 or NEP, without modifying the respective splicing and packaging signals of the viral segment. The fitness of these synthetic viruses was attenuated in vivo, while they retained immunogenicity, conferring both homologous and heterologous protection against influenza A virus challenges. These results indicate that influenza viruses can be effectively attenuated by synonymous codon deoptimization of the NS gene and open the possibility of their use as a safe vaccine to prevent infections with these important human pathogens. Vaccination serves as the best therapeutic option to protect humans against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease cause by this important human respiratory pathogen. The nonstructural (NS) gene of influenza virus encodes both the multifunctional nonstructural protein 1 (NS1), essential for innate immune evasion, and the nuclear export protein (NEP), required for the nuclear export of viral ribonucleoproteins and for timing of the virus life cycle. Here, we have generated a recombinant influenza A/Puerto Rico/8/1934 (H1N1) (PR8) virus containing a codon-deoptimized NS segment that is attenuated in vivo yet retains immunogenicity and protection efficacy against homologous and heterologous influenza virus challenges. These results open the exciting possibility of using this NS codon deoptimization methodology alone or in combination with other approaches for the future development of vaccine candidates to prevent influenza viral infections.

A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.

Bovine lactoferrin inhibits Influenza A virus induced programmed cell death in vitro

Influenza is one of the main plagues worldwide. The statistical likelihood of a new pandemic outbreak, together with the alarming emergence of influenza virus strains that are resistant to available antiviral medications, highlights the need for new antiviral drugs. Lactoferrin, a 80 kDa bi-globular iron-binding glycoprotein, is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Although the antiviral effect of lactoferrin is one of its major biological functions, the mechanism of action is still under debate. In this research, we have analyzed the effect of bovine lactoferrin (bLf) on Influenza A virus infection in vitro. Our results showed that (i) Influenza virus infected cells died as a result of apoptosis, (ii) bLf treatment inhibited programmed cell death by interfering with function of caspase 3, a major virus-induced apoptosis effector, and (iii) bLf efficiently blocked nuclear export of viral ribonucleoproteins so preventing viral assembly. These results provide further insights on the antiviral activity of bLf and suggest novel strategies for treatment of Influenza virus infection.

Acetylsalicylic acid (ASA) blocks influenza virus propagation via its NF-?B-inhibiting activity

Influenza is still one of the major plagues worldwide. The statistical likeliness of a new pandemic outbreak highlights the urgent need for new and amply available antiviral drugs. We and others have shown that influenza virus misuses the cellular IKK/NF-kappaB signalling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here we examined acetylsalicylic acid (ASA), also known as aspirin, a widely used drug with a well-known capacity to inhibit NF-kappaB. We show that the drug efficiently blocks influenza virus replication in vitro and in vivo in a mechanism involving impaired expression of proapoptotic factors, subsequent inhibition of caspase activation as well as block of caspase-mediated nuclear export of viral ribonucleoproteins. As ASA showed no toxic side-effects or the tendency to induce resistant virus variants, existing salicylate-based aerosolic drugs may be suitable as anti-influenza agents. This is the first demonstration that specific targeting of a cellular factor is a suitable approach for anti-influenza virus intervention.

The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C

The M1 protein of influenza A virus has multiple regulatory functions during the infectious cycle, which include mediation of nuclear export of viral ribonucleoproteins, inhibition of viral transcription and a crucial role in virus assembly and budding. The only known modification of the M1 protein is by phosphorylation through yet-to-be-identified kinases. We postulated that at least some of the M1 functions are exerted or regulated through interactions with cellular components. In a screen for such cellular mediators, the protein receptor of the activated C-kinase (RACK 1) was identified by its interaction with the viral M1 protein in the yeast two hybrid system. The physical M1–RACK 1 interaction was confirmed in glutathione-S-transferase-based coprecipitation assays for the diverged M1 proteins of avian, swine and human influenza A virus strains. This conservation suggests that the M1–RACK 1 interaction is of general importance during influenza A virus infections. RACK 1 has previously been identified to specifically bind the activated form of protein kinase C (PKC) and is assumed to anchor the kinase at membranes in the vicinity of its substrates. Since the M1 protein becomes phosphorylated during influenza virus infection, we examined if PKC could catalyze the phosphate transfer. We demonstrate that virion-derived and recombinant M1 protein can indeed be efficiently phosphorylated by purified PKC. Moreover, in cell extracts, we detected M1 phosphorylation activity that was strongly reduced in the presence of the PKC-specific inhibitor compound GF109203X. These data suggest that PKC is the main M1-phosphorylating activity in the cell. Since both, the M1 protein and PKC have been shown to interact with RACK 1, we suggest that the M1–RACK 1 interaction is involved in M1 phosphorylation.

Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins.

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 factor.

Viruses, microorganisms and scientists meet the nuclear pore. Leysin, VD, Switzerland, February 26-March 1, 1998.

The modular architecture of the nuclear pore complex (NPC) consists of a membrane‐embedded basic framework (the ‘spoke complex’) made up of eight multidomain spokes with two rings on each face (Figure 1A). The ring facing the cytoplasm is decorated with eight ∼50 nm fibrils extending into the cytosol, and the nuclear ring is capped with a basket‐like assembly of eight thin, 50–100 nm filaments joined distally by a 30–40 nm diameter terminal ring. The center of the basic framework harbors the ‘gated transport channel’, which is involved in signal‐mediated bidirectional transport of macromolecules and is plugged to the central ‘transporter’, whose ultimate structure and functional role in mediated nuclear transport remains to be established. N.Pante and U.Aebi (Basel) reported their approaches to the functional roles of the different NPC components and to dissection of the steps of nuclear import and export at the NPC by direct visualization of gold‐labeled proteins and RNAs moving in or out of the cell nucleus through the NPC (Figure 1B). Figure 1. ( A ) Current consensus model of the membrane‐bound NPC. Its major structural components include the basic framework, the central plug or gated channel, the cytoplasmic and nuclear rings, and the cytoplasmic fibrils and nuclear basket. ( B ) Visualization of the different steps of nuclear import of an NLS‐bearing protein through the NPC. At the bottom are selected examples of cross‐sectioned NPCs with associated nucleoplasmin–gold particles (8 nm diameter), depicted at different stages of transport; at the top is a schematic diagram of the distinct steps visualized by electron microscopy. In the first step of transport, the NLS protein to be imported associates with the NLS receptor (importins α and β) (1). This step takes place in the cytoplasm and does not require physical interaction with any NPC component. In a second step, this ‘targeting complex’ docks …

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.

Cell-to-cell movement of plant viruses. Insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems.

SummaryCell-to-cell movement is a crucial step in plant virus infection. In many viruses, the movement function is secured by specific virus-encoded proteins. Amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. This superfamily combines proteins of viruses belonging to all principal groups of positive-strand RNA viruses, as well as single-stranded DNA containing geminiviruses, double-stranded DNA-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand RNA genomes with two ambisense segments. In several groups of positive-strand RNA viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative RNA helicase. A distinct type of movement proteins with very high content of proline is found in tymoviruses. It is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. Recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. Limited sequence similarities were observed between i) movement proteins of dianthoviruses and the MIP family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and M1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. It is hypothesized that all movement proteins of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules.

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.