Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain.

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDFGREGYS). It was also shown that the SGYDFEREGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

EVOLUTIONARY DYNAMICS OF STRUCTURAL AND FUNCTIONAL DOMAINS OF INFLUENZA A VIRUS NS1 PROTEIN.

Influenza A virus (IAV) NS1 protein is one of the key viral factors responsible for virus-host interactions. NS1 counteracts host antiviral defense, participates in the processing and export of cellular mRNAs, regulates the activity of viral RNA polymerase and the expression of viral genes, and influences the cellular signaling systems. Multiple NS1 functions are carried out due to the interactions with cellular factors, the number of which exceeds one hundred. It is noteworthy that only two segments of IAV genome - NS and NP - did not undergo reassortment and evolved in the course of genetic drift, beginning with the pandemic of 1918 to the present. This fact may indicate the importance of NS1 and its numerous interactions with cellular factors in the interspecific adaptation of the virus. The review presents data on the evolution of the human IAV NS1 protein and analysis of the amino acid substitutions in the main structural and functional domains of NS1 protein during evolution.

COMPARISON OF INFLUENZA A VIRUS INHIBITION IN VITRO BY SIRNA COMPLEXES WITH CHITOSAN DERIVATIVES, POLYETHYLENEIMINE AND HYBRID POLYARGININE-INORGANIC MICROCAPSULES.

Anti-influenza drugs and vaccines have a limited effect due to the high mutation rate of virus genome. The direct impact on the conservative virus genome regions should significantly improve therapeutic effectiveness. The RNA interference mechanism (RNAi) is one of the modern approaches used to solve this problem. In this work, we have investigated the antiviral activity of small interfering RNA (siRNA) against the influenza A/PR/8/34 (H1N1), targeting conserved regions of NP and PA. Polycations were used for intracellular siRNA delivery: chitosan's derivatives (methylglycol and quaternized chitosan), polyethyleneimine, lipofectamine, and hybrid organic/non-organic microcapsules. A comparative study of these delivery systems with fluorescent labeled siRNA was conducted. The antiviral activity of three small interfering RNAs targeting the NP (NP-717, NP-1496) and PA (PA-1630) influenza A viruses genes was demonstrated, depending on the chosen carrier. The most effective intracellular delivery and antiviral activity were observed for hybrid microcapsules.

The influenza A virus NS genome segment displays lineage-specific patterns in predicted RNA secondary structure.

BACKGROUND: Influenza A virus (IAV) is a segmented negative-sense RNA virus that causes seasonal epidemics and periodic pandemics in humans. Two regions (nucleotide positions 82-148 and 497-564) in the positive-sense RNA of the NS segment fold into a multi-branch loop or hairpin structures. RESULTS: We studied 25,384 NS segment positive-sense RNA unique sequences of human and non-human IAVs in order to predict secondary RNA structures of the 82-148 and 497-564 regions using RNAfold software, and determined their host- and lineage-specific distributions. Hairpins prevailed in avian and avian-origin human IAVs, including H1N1pdm1918 and H5N1. In human and swine IAV hairpins distribution varied between evolutionary lineages. CONCLUSIONS: These results suggest a possible functional role for these RNA secondary structures and the need for experimental evaluation of these structures in the influenza life cycle.

INFLUENCE OF INFLUENZA A VIRUS AND BACTERIAL LIPOPOLYSACCHARIDE ON PROLIFERATION AND GENE EXPRESSION OF CYTOKINES AND OTHER CELLULAR FACTORS IN CELLS OF ESTABLISHED ENDOTHELIAL CELL LINE ECV-304.

Change of state of endothelial cells occurs under the action of viral infection and bacterial lipopolysaccharide (LPS) that leads to cell dysfunction. Therefore, the aim of the current study was to investigate the effect of LPS from Escherichia coli and influenza A virus on proliferative activity of human endothelial cells (ECV-304) and gene expression of several cytokines and cellular factors: TNFá, TGFâ, IFN-ã, MMP-9, NF-êB, Rho A, eNOS and iNOS. It was found that ECV-304 cells once infected with very low infectious doses of influenza virus acquire the ability to long-term active proliferation (over 8 passages). Addition of LPS E. coli reduced the virus-stimulated cell proliferation. It was shown that influenza virus and LPS can affect on gene expression of cytokine and other cellular factors. When endothelial cells had been infected with influenza A virus in the presence of LPS, there was a significant increase in the expression of several genes and replacement of some genes expression on the expression of other genes. Expression of MMP-9 gene was inhibited in the case of separate exposure to the virus and LPS, but it was significantly increased during the first day under the adding of the virus and LPS together, as well as the activity of the IFN-ã gene; gene of TNFá was active for only 1­3 days whereas genes expression of other factors (TGFâ, eNOS, iNOS, NF-êB and Rho A) increased significantly at the 5th day as in the case of adding only LPS. Thus, the change of physiological state of endothelial cells occurs in the presence of influenza A virus and LPS and it can be caused during different time periods (as well as by varying degrees of virus infection of cells) by different cellular factors and possibly with involvement of different signaling pathways.

Molecular mechanisms enhancing the proteome of influenza A viruses: an overview of recently discovered proteins.

Influenza A virus is one of the major human pathogens. Despite numerous efforts to produce absolutely effective anti-influenza drugs or vaccines, no such agent has been developed yet. One of the main reasons for this complication is the high mutation rate and the specific structure of influenza A viruses genome. For more than 25 years since the first mapping of the viral genome, it was believed that its 8 genome segments encode 10 proteins. However, the proteome of influenza A viruses has turned out to be much more complex than previously thought. In 2001, the first accessory protein, PB1-F2, translated from the alternative open reading frame, was discovered. Subsequently, six more proteins, PB1-N40, PA-X, PA-N155, PA-N182, M42, and NS3, have been found. It is important to pay close attention to these novel proteins in order to evaluate their role in the pathogenesis of influenza, especially in the case of outbreaks of human infections with new avian viruses, such as H5N1 or H7N9. In this review we summarize the data on the molecular mechanisms used by influenza A viruses to expand their proteome and on the possible functions of the recently discovered viral proteins.

[Universal diagnostic oligonucleotide microarray for subtyping of human and animal influenza A viruses].

The diagnostic oligonucleotide microarray for subtyping of human and animal influenza A viruses (IAVs) was developed. We proposed a simple method of the fluorescent labeling of genomic segments of all known IAVs subtypes, the composition of the hybridization buffer, as well as the software of the data processing. 48 IAVs strains of different subtypes were analyzed using our microarray. All of them were identified, while 45 of 48 strains were unambiguously subtyped.