Visualizing the Beta Interferon Response in Mice during Infection with Influenza A Viruses Expressing or Lacking Nonstructural Protein 1

The innate host defense against influenza virus is largely dependent on the type I interferon (IFN) system. However, surprisingly little is known about the cellular source of IFN in the infected lung. To clarify this question, we employed a reporter mouse that contains the firefly luciferase gene in place of the IFN-β-coding region. IFN-β-producing cells were identified either by simultaneous immunostaining of lungs for luciferase and cellular markers or by generating conditional reporter mice that express luciferase exclusively in defined cell types. Two different strains of influenza A virus were employed that either do or do not code for nonstructural protein 1 (NS1), which strongly suppresses innate immune responses of infected cells. We found that epithelial cells and lung macrophages, which represent the prime host cells for influenza viruses, showed vigorous IFN-β responses which, however, were severely reduced and delayed if the infecting virus was able to produce NS1. Interestingly, CD11c⁺ cell populations that were either expressing or lacking macrophage markers produced the bulk of IFN-β at 48 h after infection with wild-type influenza A virus. Our results demonstrate that the virus-encoded IFN-antagonistic factor NS1 disarms specifically epithelial cells and lung macrophages, which otherwise would serve as main mediators of the early response against infection by influenza virus.     

Generation of High-yield Vaccine Strain Wholly Derived from Avian Influenza Viruses by Reverse Genetics

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the haemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 were generated by cell transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with haemagglutination unit of 1:2 048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce haemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and the maximum geometric mean HI-titers were observed 4–5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1-infected and H5N3-vaccinated animals.     

Toscana Virus NSs Protein Inhibits the Induction of Type I Interferon by Interacting with RIG-I

Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71–79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.     

In Vitro Translation of Uukuniemi Virus-Specific RNAs: Identification of a Nonstructural Protein and a Precursor to the Membrane Glycoproteins

We isolated the virus-specific RNA species from Uukuniemi virus-infected chicken embryo cells and fractionated them by sucrose gradient centrifugation. In addition to three RNA species cosedimenting with the three viral RNA segments L (29S), M (23S), and S (17S), a fourth major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of both plus and minus strands, with the plus strands in slight excess. Most of the S1 RNA was of negative polarity, whereas S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000-dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a molecular weight of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. It is thus likely that the glycoproteins are synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent molecular weight of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.     

Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.     

Nuclear Trafficking of the Rabies Virus Interferon Antagonist P-Protein Is Regulated by an Importin-Binding Nuclear Localization Sequence in the C-Terminal Domain

Rabies virus P-protein is expressed as five isoforms (P1-P5) which undergo nucleocytoplasmic trafficking important to roles in immune evasion. Although nuclear import of P3 is known to be mediated by an importin (IMP)-recognised nuclear localization sequence in the N-terminal region (N-NLS), the mechanisms underlying nuclear import of other P isoforms in which the N-NLS is inactive or has been deleted have remained unresolved. Based on the previous observation that mutation of basic residues K214/R260 of the P-protein C-terminal domain (P-CTD) can result in nuclear exclusion of P3, we used live cell imaging, protein interaction analysis and in vitro nuclear transport assays to examine in detail the nuclear trafficking properties of this domain. We find that the effect of mutation of K214/R260 on P3 is largely dependent on nuclear export, suggesting that nuclear exclusion of mutated P3 involves the P-CTD-localized nuclear export sequence (C-NES). However, assays using cells in which nuclear export is pharmacologically inhibited indicate that these mutations significantly inhibit P3 nuclear accumulation and, importantly, prevent nuclear accumulation of P1, suggestive of effects on NLS-mediated import activity in these isoforms. Consistent with this, molecular binding and transport assays indicate that the P-CTD mediates IMPα2/IMPβ1-dependent nuclear import by conferring direct binding to the IMPα2/IMPβ1 heterodimer, as well as to a truncated form of IMPα2 lacking the IMPβ-binding autoinhibitory domain (ΔIBB-IMPα2), and IMPβ1 alone. These properties are all dependent on K214 and R260. This provides the first evidence that P-CTD contains a genuine IMP-binding NLS, and establishes the mechanism by which P-protein isoforms other than P3 can be imported to the nucleus. These data underpin a refined model for P-protein trafficking that involves the concerted action of multiple NESs and IMP-binding NLSs, and highlight the intricate regulation of P-protein subcellular localization, consistent with important roles in infection.     

Mutant Influenza Viruses with a Defective NS1 Protein Cannot Block the Activation of PKR in Infected Cells

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5′- and 3′-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2α). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2α phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2α.     

Some Common Properties Between a Brain Protein that Is Modified by Posttranslational Arginylation and the Microtubule-Associated STOP Protein

Abstract: Properties so far studied of the 125-kDa ¹⁴C-arginylated protein from rat brain show remarkable similarities with those of the STOP (stable tubule only polypeptide) protein. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the 125-kDa ¹⁴C-arginylated protein moves to the same position as the STOP protein. The 125-kDa ¹⁴C-arginylated protein was immunoprecipitated by the monoclonal Mab 296 antibody specific for neuronal STOP protein. The 125-kDa ¹⁴C-arginylated protein was retained by a calmodulin column like STOP protein. As occurs with the STOP protein, the 125-kDa ¹⁴C-arginylated protein is found in higher proportion in cold-stable than in cold-labile microtubules. However, the modified protein associates with microtubules in a lower proportion than the STOP protein. We conclude that the STOP protein incorporates arginine by a posttranslational reaction but that only a small fraction of the STOP protein shows acceptor capacity in vitro.     

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P_BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione S-transferase–NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of ~50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.