H5N1 Nucleoprotein Research Articles

P-cymene impairs SARS-CoV-2 and Influenza A (H1N1) viral replication: In silico predicted interaction with SARS-CoV-2 nucleocapsid protein and H1N1 nucleoprotein.

Therapeutic regimens for the COVID‐19 pandemics remain unmet. In this line, repurposing of existing drugs against known or predicted SARS‐CoV‐2 protein actions have been advanced, while natural products have also been tested. Here, we propose that p‐cymene, a natural monoterpene, can act as a potential novel agent for the treatment of SARS‐CoV‐2‐induced COVID‐19 and other RNA‐virus‐induced diseases (influenza, rabies, Ebola). We show by extensive molecular simulations that SARS‐CoV‐2 C‐terminal structured domain contains a nuclear localization signal (NLS), like SARS‐CoV, on which p‐cymene binds with low micromolar affinity, impairing nuclear translocation of this protein and inhibiting viral replication, as verified by preliminary in vitro experiments. A similar mechanism may occur in other RNA‐viruses (influenza, rabies and Ebola), also verified in vitro for influenza, by interaction of p‐cymene with viral nucleoproteins, and structural modification of their NLS site, weakening its interaction with importin A. This common mechanism of action renders therefore p‐cymene as a possible antiviral, alone, or in combination with other agents, in a broad spectrum of RNA viruses, from SARS‐CoV‐2 to influenza A infections.

Crystal structures of influenza nucleoprotein complexed with nucleic acid provide insights into the mechanism of RNA interaction.

The nucleoprotein (NP) of influenza virus is the core component of the ribonucleoprotein (RNP) and performs multiple structural and functional roles. Structures of the influenza A, B and D NP molecules have been solved previously, but structural information on how NP interacts with RNA remains elusive. Here we present the crystal structure of an obligate monomer of H5N1 NP in complex with RNA nucleotides to 2.3 Å, and a C-terminal truncation of this mutant, also in complex with RNA nucleotides, to 3 Å. In both structures, three nucleotides were identified near two positive grooves of NP suggested to be important for RNA binding. Structural evidence supports that conformational changes of flexible loops and the C-terminal tail both play important roles in the binding of RNA. Based on the structure, we propose a mechanism by which NP captures RNA by flexible loops and transfers it onto the positive binding grooves. Binding of RNA by NP is a crucial step for template re-encapsidation during transcription and replication and cRNP formation. Our structures thus provide insights into the molecular virology of the influenza virus.

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

BackgroundProtection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising.MethodsThis study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection.ResultsIFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry.ConclusionsThis study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

An integrative docking and simulation-based approach towards the development of epitope-based vaccine against enterotoxigenic Escherichia coli.

Enterotoxigenic E.coli is causing diarrheal illness in children as well as adults with the majority of the cases occurring in developing countries. To reduce the number of cases occurring worldwide, the development of an effectual vaccine against these bacteria can be the only prevention. This conjectural work was performed using modern bioinformatics tools for investigation of proteome of ETEC strain E24377A. Different computational vaccinology approaches were deployed to assess several parameters including antigenicity, allergenicity, stability, localization, molecular weight and toxicity of the predicted epitopes required for good vaccine candidate to elicit immune response against diarrhea. We estimated two known control antigens, epitope 141STLPETTVV149 of Hepatitis B virus and epitope 265ILRGSVAHK273 of H1N1 Nucleoprotein in an attempt to corroborate our research work. Furthermore molecular docking was performed to evaluate the interaction between HLA allele and peptide, the peptide QYGGGNSAL and peptide LPYFELRWL were considered to be the most promiscuous T cell epitopes with the highest binding energy value of −2.09 kcal/mol and −1.84 kcal/mol, respectively. In addition, dynamic simulation revealed good stability of the vaccine construct as well as population coverage analysis exhibits the highest population coverage in the regions of East Asia, India, Northeast Asia, South Asia and North America. Therefore, these two epitopes can be further synthesized for wet lab analysis and could be considered as a promising vaccine against diarrhea.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13721-021-00287-6.

Magnetic Particle Spectroscopy for Detection of Influenza A Virus Subtype H1N1.

Magnetic nanoparticles (MNPs) with proper surface functionalization have been extensively applied as labels for magnetic immunoassays, carriers for controlled drug/gene delivery, tracers and contrasts for magnetic imaging, etc. Here, we introduce a new biosensing scheme based on magnetic particle spectroscopy (MPS) and the self-assembly of MNPs to quantitatively detect H1N1 nucleoprotein molecules. MPS monitors the harmonics of oscillating MNPs as a metric for the freedom of rotational process, thus indicating the bound states of MNPs. These harmonics can be readily collected from nanogram quantities of iron oxide nanoparticles within 10 s. The H1N1 nucleoprotein molecule hosts multiple different epitopes that forms binding sites for many IgG polyclonal antibodies. Anchoring IgG polyclonal antibodies onto MNPs triggers the cross-linking between MNPs and H1N1 nucleoprotein molecules, thereby forming MNP self-assemblies. Using MPS and the self-assembly of MNPs, we were able to detect as low as 44 nM (4.4 pmole) H1N1 nucleoprotein. In addition, the morphologies and the hydrodynamic sizes of the MNP self-assemblies are characterized to verify the MPS results. Different MNP self-assembly models such as classical cluster, open ring tetramer, and chain model as well as multimers (from dimer to pentamer) are proposed in this paper. Herein, we claim the feasibility of using MPS and the self-assembly of MNPs as a new biosensing scheme for detecting ultralow concentrations of target biomolecules, which can be employed as rapid, sensitive, and wash-free magnetic immunoassays.

(-)-Epigallocatechin-3-gallate induces interferon-λ2 expression to anti-influenza A virus in human bronchial epithelial cells (BEAS-2B) through p38 MAPK signaling pathway.

Background(-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea, has been found to inhibit the influenza virus. However, the mechanism of EGCG anti-influenza virus effect needs to be further explored.MethodsBEAS-2B cells were treated with different concentrations of EGCG or were treated with EGCG for different times. CCK8 assay was used to detect the cell viability, and quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay were employed to measure the interferon (IFN)-λ2 mRNA and protein expression levels. The phospho-p38 mitogen-activated protein kinase (P-p38 MAPK), phospho-extracellular signal-regulated kinase (P-ERK), and phospho-c-Jun N-terminal kinase (P-JNK) expression were tested by western blot. Then, p38 MAPK, ERK, and JNK inhibitor were used to study the effect of p38 MAPK, ERK, and JNK signaling pathways on IFN-λ2 expression. The BEAS-2B cells were treated with EGCG, EGCG and IFN λ2 neutralizing antibody or control antibody for 12 h, and were infected with influenza A virus (IAV) (H1N1) for 1 h. After 12 h, nucleoprotein (NP) mRNA and protein expression levels of H1N1 were assessed by qRT-PCR and western blot.ResultsThe IFN-λ2 mRNA and protein expression levels in BEAS-2B cells were up-regulated after EGCG (treatment in time- and dose-dependent manners the concentration range from 0 to 50 µg/mL had no cytotoxicity). Meanwhile, the P-p38 MAPK, P-ERK, and P-JNK expression levels were up-regulated. IFN-λ2 mRNA and protein expression was inhibited after p38 MAPK inhibitor pre-treatment, but not by ERK and JNK inhibitors. Furthermore, the expression of H1N1 NP gene and protein decreased after EGCG pre-treatment, while IFN-λ2 neutralizing antibody attenuated the effect of EGCG inhibiting the expression of H1N1 NP gene and protein.ConclusionsEGCG inhibited IAV H1N1 by inducing the expression of IFN-λ2 in BEAS-2B cells through the p38 MAPK signaling pathway.

Highly sensitive detection of protein biomarkers via nuclear magnetic resonance biosensor with magnetically engineered nanoferrite particles.

Magnetic-based biosensors are attractive for on-site detection of biomarkers due to the low magnetic susceptibility of biological samples. Here, we report a highly sensitive magnetic-based biosensing system that is composed of a miniaturized nuclear magnetic resonance (NMR) device and magnetically engineered nanoferrite particles (NFPs). The sensing performance, also identified as the transverse relaxation (R2) rate, of the NMR device is directly related to the magnetic properties of the NFPs. Therefore, we developed magnetically engineered NFPs (MnMg-NFP) and used them as NMR agents to exhibit a significantly improved R2 rate. The magnetization of the MnMg-NFPs was increased by controlling the Mn and Mg cation concentration and distribution during the synthesis process. This modification of the Mn and Mg cation directly contributed to improving the R2 rate. The miniaturized NMR system, combined with the magnetically engineered MnMg-NFPs, successfully detected a small amount of infectious influenza A H1N1 nucleoprotein with high sensitivity and stability.

Highly Pathogenic Avian Influenza Virus Nucleoprotein Interacts with TREX Complex Adaptor Protein Aly/REF

We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

Structural and Functional Characterization of K339T Substitution Identified in the PB2 Subunit Cap-binding Pocket of Influenza A Virus

Influenza virus RNA-dependent RNA polymerase is a heterotrimer composed of PA, PB1, and PB2 subunits. RNA-dependent RNA polymerase is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A "cap-snatching" mechanism is used to generate a 5'-capped primer for transcription in which the cap-binding domain of PB2 (PB2cap) captures the 5' cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys(339) located in the cap-binding pocket of H5N1 PB2cap was gradually replaced by Thr(339) over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2cap with or without a pre-mRNA cap analog, m(7)GTP, in the presence of Lys(339) or Thr(339). The structures showed that Lys(339) contributes to binding the γ-phosphate group of m(7)GTP, and the replacement of Lys(339) by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr(339) attenuated the PB2cap cap binding activity in vitro compared with Lys(339). Further functional studies confirmed that Thr(339)-PB2-containing ribonucleoprotein complex has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr(339) substitution exhibited a lower virulence to mice but more active replication in Madin-Darby canine kidney cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.

Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding.

The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73–90), loop 2 (200–214). In NP, loops 1 and 2 formed a 10–15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91–112) that interacted with the RNA grove (linker 360–373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein.

Functional Analysis of the Influenza Virus H5N1 Nucleoprotein Tail Loop Reveals Amino Acids That Are Crucial for Oligomerization and Ribonucleoprotein Activities

Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.

Purification and production of monospecific antibody to the hemagglutinin from subtype H5N1 influenza virus.

The purpose of this study was to purify the hemagglutinin from H5N1 virus and to generate monospecific antibody appropriate for production of sensitive and specific immunoassay for H5N1 avian influenza. For this purpose, a local isolate H5N1 virus (A/Ck/West Java/Hamd/2006) was propagated in chicken embryos. The viral pellet was dissolved in a Triton-X-100 solution, undissolved viral particles were pelleted by ultracentrifuge, and the supernatant containing viral surface glycoproteins (Hemagglutinin and neuraminidase) was collected. The neuraminidase in the supernatant was absorbed by passing the supernatant through an Oxamic-acid-superose column. After dialyzing extensively, the filtrate was further fractionated with an anion exchange chromatography (Q-sepharose) column. Proteins adsorbed by the column were eluted stepwisely with 0.10, 0.25, 0.25 and 0.75 M NaCl in 20 m M Tris, ph 8. Hemagglutinin (H5) was found to be eluted from the column with the 0.5 M NaCl elution buffer. The purified H5 was free from other viral proteins based on immunoassays using commercial antibodies to H5N1 nucleoprotein and neuraminidase. When used as ELISA’s coating antigen, the purified H5 proved to be sensitive and specific for hemagglutinin H5. Cross reactions with other type-A-influenza virus, H6, H7 dan H9, were negligibly low. For the production of monospecific antiserum, the purified H5 was separated with SDS-PAGE, the band containing the H5 monomer was cut out , homogenised and injected into rabbits. The antiserum was capable of detecting the presence of inactivated H5N1 virus in a very dilute suspension, with a detection limit of 0.04 heagglutination (HA) unit. The purified hemagglutinin and the serum raised against it should be useful for developing specific, sensitive and affordable immunoassay for H5N1 avian influenza. Key Words : H5N1, Hemagglutinin, Triton, Oxamic-acid Sepharose, Q sepharose, ELISA

Diagnostic Assay Recommended by the World Health Organization for Swine Origin Influenza A (H1N1) Virus Cross-Reacts with H5N1 Influenza Virus

Pandemic influenza poses a significant risk to global human health. The unpredictable nature of the influenza virus necessitates accurate, reliable, and rapid diagnostic assays for the detection of, and characterization of the current pandemic caused by, swine origin influenza virus (SOIV) in order to better understand the nature of the pandemic and implement appropriate health interventions. SOIV emerged in mid-April 2009 (1), leaving laboratories around the world scrambling to establish a diagnostic test to detect this novel influenza virus. The World Health Organization (WHO) responded with remarkable speed by releasing guidelines and protocols for a real-time reverse transcription (RT)-PCR assay 15 days after the reported identification of SOIV (2). This provided a rapid and sensitive assay for the detection of SOIV that was critical for the determination of the spread and extent of SOIV infections. These guidelines recommend three primer-and-probe sets: InfA, amplifying a conserved region of the matrix gene from all influenza A viruses; SW H1, designed to specifically detect the hemagglutinin gene segment (subtype H1) from SOIV; and SW InfA, designed to specifically detect the nucleoprotein (NP) gene segment from all swine influenza viruses. As part of the WHO Global Influenza Surveillance Network, the New Zealand National Influenza Centre regularly participates in WHO external quality assurance panels of 10 RNA samples that are H1N1, H3N2, or H5N1 influenza A virus or, recently, SOIV. In following the WHO recommended guidelines exactly, it was found that the SW InfA primer-and-probe set amplified all six H5N1 RNA samples. This is clinically relevant, as the proficiency panel RNA returned threshold cycle (CT) values (InfA assay, 23.44 to 28.67) well within the range of what we regularly observe with clinical samples. In addition, the SW InfA primer-and-probe set detected two additional H5N1 viruses not provided in the proficiency panel, the human H5N1 strain A/Vietnam/3028/2004 and an avian H5N1 strain, A/Mallard/New Zealand/272-73/2008. The efficiency of this reaction was 79% (three replicates) compared to 94% (five replicates) for the amplification of SOIV RNA, suggesting that mismatches within the primer target region reduced assay efficiency. To prove that the H5N1 NP gene sequence was amplified by the SOIV SW InfA assay, all real-time RT-PCR amplicons were sequenced. The two SOIV RNA samples from the proficiency panel and SOIV-positive control showed 100% identity to the consensus sequence of 394 NP full-length gene segments available as of 29 July 2009. In contrast, all H5N1 RNA samples from the proficiency panel contained at least 31 nucleotide differences from the SOIV consensus sequence but showed between 93.3% and 96.8% identity to A/Vietnam/1203/2004. Alignment of all SOIV and human H5N1 NP gene segments available in GenBank showed that 391 SOIV viruses have one mismatch in the SW InfA forward primer (Table ​(Table1).1). The remaining three SOIV sequences have a further mismatch in either the forward primer or the probe. The majority (74.6%, or 91 sequences) of available human H5N1 sequences are identical within the SW InfA primer-and-probe regions, having two mismatches in the forward primer, two in the probe, and four in the reverse primer region. None of the mismatches occurred within 9 nucleotides of the 3′ end, an important determinant for primer specificity. TABLE 1. Comparison of SOIV and human H5N1 NP gene sequences with SW infA primer and probe sequences The speed at which these assays were developed and deployed to the WHO Global Influenza Surveillance Network was remarkable, particularly with the limited SOIV sequence information available at the time of development. Other factors which constrained the development of RT-PCR primer targets include the origin and genetic rate of change for each gene segment; only five of the eight gene segments are of swine origin (3), while the rapidly evolving HA and NA genes have a propensity to drift at a much higher rate than conserved segments such as NP (5). This report demonstrates that the SW InfA assay is not specific to SOIV and is able to detect both human and avian (H5N1) influenza A viruses and so there is the potential for misidentification. Following WHO guidelines, the utilization of all primer-and-probe sets for the detection of SOIV should provide an accurate diagnosis. However, we have observed that for the same SOIV RNA sample, the SW InfA assay consistently returns lower CT values than the SW H1 assay and so clinical samples with higher CT values can yield an SW H1-negative, SW InfA-positive result. In these cases, we suggest sequencing of real-time SW InfA PCR amplicons to ensure the amplification of SOIV (4). Alternatively, and especially for countries in which H5N1 is endemic, an H5N1-specific assay could be performed to exclude this diagnosis. However, a negative H5N1 result would not exclude other influenza virus subtypes that may cross-react with the SOIV assay.

Neutrophils May Be a Vehicle for Viral Replication and Dissemination in Human H5N1 Avian Influenza

The mechanism of systemic spread of H5N1 virus in patients with avian influenza is unknown. Here, H5N1 nucleoprotein and hemagglutinin were identified by immunohistochemistry in the nucleus and cytoplasm of neutrophils in the placental blood of a pregnant woman. Viral RNA was detected in neutrophils by in situ hybridization and enhanced real-time polymerase chain reaction. Therefore, neutrophils may serve as a vehicle for viral replication and transportation in avian influenza.

Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design

The threat of a pandemic outbreak of influenza virus A H5N1 has become a major concern worldwide. The nucleoprotein (NP) of the virus binds the RNA genome and acts as a key adaptor between the virus and the host cell. It, therefore, plays an important structural and functional role and represents an attractive drug target. Here, we report the 3.3-A crystal structure of H5N1 NP, which is composed of a head domain, a body domain, and a tail loop. Our structure resolves the important linker segments (residues 397-401, 429-437) that connect the tail loop with the remainder of the molecule and a flexible, basic loop (residues 73-91) located in an arginine-rich groove surrounding Arg150. Using surface plasmon resonance, we found the basic loop and arginine-rich groove, but mostly a protruding element containing Arg174 and Arg175, to be important in RNA binding by NP. We also used our crystal structure to build a ring-shaped assembly of nine NP subunits to model the miniribonucleoprotein particle previously visualized by electron microscopy. Our study of H5N1 NP provides insight into the oligomerization interface and the RNA-binding groove, which are attractive drug targets, and it identifies the epitopes that might be used for universal vaccine development.

Detection of mouse-adapted human influenza virus in the olfactory bulbs of mice within hours after intranasal infection

Influenza pneumonitis causes severe systemic symptoms in mice, including hypothermia and excess sleep. The association of extrapulmonary virus, particularly virus in the brain, with the onset of such disease symptoms has not been investigated. Mature C57BL/6 male mice were infected intranasally with mouse-adapted human influenza viruses (PR8 or X-31) under inhalation, systemic, or no anesthesia. Core body temperatures were monitored continuously by radiotelemetry, and tissues (lung, brain, olfactory bulb, spleen, blood) were harvested at the time of onset of hypothermia (13 to 24 h post infection [PI]) or at 4 or 7 h PI. Whole RNA from all tissues was examined by one or more of three reverse transcriptase-polymerase chain reaction (RT-PCR) procedures using H1N1 nucleoprotein (NP) primers for minus polarity RNA (genomic or vRNA) or plus polarity RNA (replication intermediates). Selected cytokines were assayed at 4, 7, and 15 h in the olfactory bulb (OB). Minus and plus RNA strands were readily detected in OBs as early as 4 h PI by nested RT-PCR. Anesthesia was not required for viral invasion of the OB. Cytokine mRNAs were also significantly elevated in the OB at 7 and 15 h PI in infected mice. Controls receiving boiled virus expressed only input vRNA and that only in lung. Immunohistochemistry demonstrated localization of H1N1 and NP antigens in olfactory nerves and the glomerular layer of the OB. Therefore a mouse-adapted human influenza virus strain, not known to be neurotropic, was detected in the mouse OB within 4 h PI where it appeared to induce replication intermediates and cytokines.

Costimulation by B7 modulates specificity of cytotoxic T lymphocytes: a missing link that explains some bystander T cell activation.

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366-374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide-pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.