Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cDNAs in prokaryotic and eukaryotic cells

To study the biological and immunological properties of influenza virus surface glycoproteins, cDNA copies of the haemagglutinin (HA) and the neuraminidase (NA) genes of A/WSN/33 influenza virus were cloned and expressed in prokaryotic and eukaryotic cells. In Escherichia coli, maximum expression of HA is obtained only as a fusion protein in which the NH₂-terminal portion is provided by a bacterial protein (i.e. βgal or trpLE′). The HA expressed in bacteria (bacterial HA) is recognized by polyclonal anti-WSN antibodies but not by neutralizing monoclonal antibodies. The antibodies made against the bacterial HA bind to the detergent-treated viral HA, intact virus and live influenza infected cells, but fail to show either haemagglutination inhibition (HI) or virus neutralization. These results suggest that the three-dimensional structure as well as the antigenic epitopes of the bacterial HA are different from that of native viral HA. HA, expressed from cDNA in cultured animal cells, is shown to possess the structural features of the native viral HA. It is glycosylated, transported to the apical domain of the plasma membrane of polarized cells, causes haemadsorption and can induce cell to cell fusion at low pH after proteolytic cleavage. An attempt was made to define the structural features of HA required for sorting and directional transport by making chimeras with vesicular stomatitis virus G (VSV G) protein either by switching the amino terminus or the carboxy terminus of HA with that of VSV G. These chimeric proteins were translocated across the rough endoplasmic reticulum (RER) but were blocked in transport between the RER and cell membrane. These preliminary results indicate that the three-dimensional structure, in addition to specific sequences, may play a critical role in the transport process. More precise constructions are in progress to delineate the functions of the different domains of HA molecule. HA has been expressed in the lower eukaryote Saccharomyces cerevisiae (baker's yeast). The HA expressed in yeast (yeast-HA) is glycosylated and membrane-bound. It is recognized by both the polyclonal and neutralizing monoclonal antibodies made against the native HA. These results suggest that the yeast-HA retains the antigenic epitopes present in the native viral HA and therefore may be of significance in the development of a subunit vaccine. NA, the other influenza glycoprotein, has been expressed from the cloned cDNA in cultured monkey kidney cells and the expressed NA, like the native viral NA, is glycosylated and enzymatically active. Furthermore, it has been shown that the NH₂-terminus of NA, in addition to providing the anchor function, also provides the signal function for translocation across the rough endoplasmic reticulum (RER). Like HA, NA expressed from cloned cDNA is preferentially transported to the apical domain of the plasma membrane of polarized epithelial cells.     

Combination chemotherapy for influenza

The emergence of pandemic H1N1 influenza viruses in April 2009 and the continuous evolution of highly pathogenic H5N1 influenza viruses underscore the urgency of novel approaches to chemotherapy for human influenza infection. Anti-influenza drugs are currently limited to the neuraminidase inhibitors (oseltamivir and zanamivir) and to M2 ion channel blockers (amantadine and rimantadine), although resistance to the latter class develops rapidly. Potential targets for the development of new anti-influenza agents include the viral polymerase (and endonuclease), the hemagglutinin, and the non-structural protein NS1. The limitations of monotherapy and the emergence of drug-resistant variants make combination chemotherapy the logical therapeutic option. Here we review the experimental data on combination chemotherapy with currently available agents and the development of new agents and therapy targets.     

Novel druggable hot spots in avian influenza neuraminidase H5N1 revealed by computational solvent mapping of a reduced and representative receptor ensemble

The influenza virus subtype H5N1 has raised concerns of a possible human pandemic threat because of its high virulence and mutation rate. Although several approved anti-influenza drugs effectively target the neuraminidase, some strains have already acquired resistance to the currently available anti-influenza drugs. In this study, we present the synergistic application of extended explicit solvent molecular dynamics (MD) and computational solvent mapping (CS-Map) to identify putative 'hot spots' within flexible binding regions of N1 neuraminidase. Using representative conformations of the N1 binding region extracted from a clustering analysis of four concatenated 40-ns MD simulations, CS-Map was utilized to assess the ability of small, solvent-sized molecules to bind within close proximity to the sialic acid binding region. Mapping analyses of the dominant MD conformations reveal the presence of additional hot spot regions in the 150- and 430-loop regions. Our hot spot analysis provides further support for the feasibility of developing high-affinity inhibitors capable of binding these regions, which appear to be unique to the N1 strain.     

Recent advances in the discovery and development of anti-influenza drugs

Breakthrough advances in the chemotherapy of influenza have been achieved in recent years and the approval of zanamivir (Relenza^TM, Biotech Holdings) by the United States FDA in July 1999 for the treatment of type A and type B influenza marked the first new influenza treatments since the approval of rimantadine in 1993. This was followed by the approval of oseltamivir (Tamiflu^TM, Gilead Sciences) for the treatment of both type A and type B influenza in October 1999 and, subsequently, for the prevention of influenza A and B infection in adults and adolescents over the age of 13 years. Both zanamivir and oseltamivir are inhibitors of influenza neuraminidase, one of two surface glycoproteins of influenza virus and they represent a new class of anti-influenza agent. The successful launch of zanamivir and oseltamivir has prompted continued effort to discover and develop neuraminidase inhibitors with improved properties. Indeed, most of the anti-influenza drug discovery activity in recent years has been centred on neuraminidase inhibition, although efforts on improved preparation and formulation of the anti-influenza vaccine continues. This is evidenced by 22 patent applications filed between January 1998 and October 2001 concerning the chemotherapy of influenza all dealt with neuraminidase inhibition. This article will give a critical review of the recent advances in this area.     

Bioactive constituents of oleanane-type triterpene saponins from the roots of Glycyrrhiza glabra

Three new oleanane-type triterpene saponins, namely licorice-saponin M3 (1), licorice-saponin N4 (2), and licorice-saponin O4 (3), an artificial product (4), as well as five known triterpene glucuronides (5–9), were isolated from the roots of Glycyrrhiza glabra L. Their structures were established using 1D and 2D NMR spectroscopy, mass spectrometry, and by comparison with spectroscopic data reported in the literature. The inhibitory effects of the selected compounds on neuraminidase were evaluated, and the preliminary structure–activity relationship was also predicted.     

Learning from structure-based drug design and new antivirals targeting the ribonucleoprotein complex for the treatment of influenza

Introduction: Influenza viruses are a threat to human health. There are presently only two methods for treating influenza: vaccines, which require yearly updates, and two classes of antivirals that suffer with the problem of resistance by current human influenza viruses; this is especially the case with amantadine and rimantadine. Consequently, there is an urgent need for the development of new antivirals with new mechanisms of action.

Areas covered: In this review, the authors focus on viral protein domains, their associated activity and their inhibition by small molecules defined by a structure-based design with a special emphasis on the ribonucleoprotein complex and its inhibitors. Several new classes of antiviral candidates targeting viral replication through individual domains of the polymerase and the nucleoprotein (NP) have been developed through structure-based design.

Expert opinion: To date, the antivirals targeting neuraminidase are by far the most developed and potent. Antiviral candidates targeting the NP and polymerase domains are in the pipeline but their pharmacokinetics needs further studies. The recently published structures of the polymerase expand the possibilities for development of new antivirals. Combination therapies targeting conserved viral targets and new cellular proteins or exploiting drug promiscuity hold promises to fight against the emergence of resistance.     

Peramivir for the treatment of influenza

Peramivir (BioCryst Pharmaceuticals) is a novel investigational intravenous neuraminidase inhibitor that exhibits potent antiviral activity against influenza A and B viruses. Peramivir is created by a structure-based drug design and consists of a cyclopentane backbone with a positively charged guanidinyl group and lipophilic side chains. Peramivir was made available in the USA through the Emergency Investigational New Drug regulations and under an Emergency Use Authorization for hospitalized patients with known or suspected influenza during the 2009 H1N1 influenza pandemic. In trials involving ambulatory adult subjects, intravenous peramivir is safe and has a pharmacokinetic profile that supports once-daily dosing. The drug is licensed in Japan and South Korea and is currently undergoing Phase III trials in the USA. Viral resistance mechanisms to peramivir have not been fully delineated and ongoing surveillance is important. Given the serious health threat of influenza at all ages and limitations in vaccine delivery, peramivir is a promising addition to the currently limited treatment options for the treatment of severe influenza infection.     

Emergence of a novel drug resistant H7N9 influenza virus: evidence based clinical potential of a natural IFN-α for infection control and treatment

The novel avian H7N9 influenza virus has caused more than 130 human infections with 43 deaths (as of September, 2013) in China. Because of the lack of existing immunity against H7 subtype influenza viruses in the human population and the absence of a licensed commercial vaccine, antiviral drugs are critical tools for the treatment of infection with this novel H7N9. Both M2-ion channel blockers and neuraminidase inhibitors are used as antiviral drugs for influenza infections of humans. The emerging H7N9 viruses are resistant to the M2-ion channel blockers because of a S31N mutation in the M2 protein; additionally, some H7N9 isolates have gained neuraminidase R292K substitution resulting in broad resistance to neuraminidase inhibitors. In this study we report that Alferon N can inhibit wild type and 292K H7N9 viruses replication in vitro. Since Alferon N is approved for clinical use, this would allow a rapid regulatory approval process for this drug under pandemic threat.     

Anti-influenza virus activity of the ethanolic extract from Peperomia sui

Ethnopharmacological relevance

Peperomia sui Lin and Lu (Peperomia sui), a well-known Taiwanese folk medicine, has a broad range of biological effects, especially in treatment of upper respiratory tract diseases. However, no previous study has explored the activity of Peperomia sui against influenza virus infections. This study was carried out to evaluate the anti-influenza virus activity and the potential virucidal effect of the ethanolic extract of Peperomia sui (PSE).

Methods

The anti-H6N1 avian influenza viral activity of PSE against the influenza virus A/Chicken/TW/0518/2011 (H6N1) in chicken fibroblast DF-1 cells was evaluated by cell viability assay, hemagglutination assay, neuraminidase activity assay, indirect immunofluorescence assay and quantitative RT-PCR assay.

Results

PSE significantly increased the viability of cells that were infected by the H6N1 virus. PSE also suppressed the synthesis of viral nucleoprotein (NP), and inhibited the growth of the virus in DF-1 cells. Further, PSE inhibited the neuraminidase activity of H6N1 virus.

Conclusions

The findings of this study provide important information for the exploitation and utilization of Peperomia sui in treatment of influenza infection.