Comparative analysis between a low pathogenic and a high pathogenic influenza H5 hemagglutinin in cell entry

Avian influenza viruses continue to threaten globally with pandemic potential. The first step in a potential pandemic is the ability of the virus to enter human cells which is mediated by the viral surface glycoprotein hemagglutinin (HA). Viral entry of influenza is dependent upon the processing of the HA0 polypeptide precursor protein into HA1 and HA2 which is mediated by host cellular proteases. The sequence of the cleavage site which is recognized by host proteases has been linked with pathogenesis of various influenza viruses. Here we examined the effects of cleavage site sequences between a highly pathogenic H5N1 strain and a low pathogenic H5N2 strain to determine their effects on viral entry. From this analysis we determined that at the level of viral entry, the only observed difference between the low and high pathogenic strains is their ability to be cleaved by host cellular proteases

Analysis of hemagglutinin-mediated entry tropism of H5N1 avian influenza

<p>Abstract</p> <p>Background</p> <p>Avian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to α2,3-linked SAs, while human influenza A viruses exhibit a preference for α2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses.</p> <p>Results</p> <p>We demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both α2,6-linked and α2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding.</p> <p>Conclusion</p> <p>Our results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.</p

Influenza A Virus Entry: Implications in Virulence and Future Therapeutics

Influenza A viruses have broad host tropism, being able to infect a range of hosts from wild fowl to swine to humans. This broad tropism makes highly pathogenic influenza A strains, such as H5N1, potentially dangerous to humans if they gain the ability to jump from an animal reservoir to humans. How influenza A viruses are able to jump the species barrier is incompletely understood due to the complex genetic nature of the viral surface glycoprotein, hemagglutinin, which mediates entry, combined with the virus\u27s ability to use various receptor linkages. Current therapeutics against influenza A include those that target the uncoating process after entry as well as those that prevent viral budding. While there are therapeutics in development that target entry, currently there are none clinically available. We review here the genetics of influenza A viruses that contribute to entry tropism, how these genetic alterations may contribute to receptor usage and species tropism, as well as how novel therapeutics can be developed that target the major surface glycoprotein, hemagglutinin

Viral entry and beyond: Discovery and therapeutics of influenza and HIV.

Viral entry and beyond: Discovery and therapeutics of influenza and HIV

Development of an Antiviral Screening Protocol: One-Stone-Two-Birds

As prophylactic therapies and vaccines against viral infections continue to improve, drug resistant strains are continuing to arise; therefore it is imperative to develop new therapeutics against these diseases. For highly pathogenic viruses, such as Ebola and H5N1 influenza virus, the need for antivirals is even more urgent due to limited therapeutics against these viruses. Furthermore, the high pathogenicity of such viruses often makes it difficult to work with such agents. In this report, we describe a protocol called “One-stone-two-birds” which provides a safe and efficient screening system to identify anti-flu (entry) and anti-HIV (replication) activities. Using plant extracts as an example, we demonstrate the utility of this protocol in antiviral screening

Development of an Antiviral Screening Protocol: One-Stone-Two-birds

As prophylactic therapies and vaccines against viral infections continue to improve, drug resistant strains are continuing to arise; therefore it is imperative to develop new therapeutics against these diseases. For highly pathogenic viruses, such as Ebola and H5N1 influenza virus, the need for antivirals is even more urgent due to limited therapeutics against these viruses. Furthermore, the high pathogenicity of such viruses often makes it difficult to work with such agents. In this report, we describe a protocol called “One-stone-two-birds” which provides a safe and efficient screening system to identify anti-flu (entry) and anti-HIV (replication) activities. Using plant extracts as an example, we demonstrate the utility of this protocol in antiviral screening

A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre–messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy

Characterization of Marburg Virus Glycoprotein in Characterization of Marburg Virus Glycoprotein in Viral Entry

One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289–501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP