Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir ...treatment. Methods. Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed. Results. The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir. Conclusions. Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.
There are 15 subtypes of influenza A virus (H1–H15), all of which are found in avian species. Three caused pandemics in the last century: H1 in 1918 (and 1977), H2 in 1957 and H3 in 1968. In 1997, an ...H5 avian virus and in 1999 an H9 virus caused outbreaks of respiratory disease in Hong Kong. We have determined the three‐dimensional structures of the haemagglutinins (HAs) from H5 avian and H9 swine viruses closely related to the viruses isolated from humans in Hong Kong. We have compared them with known structures of the H3 HA from the virus that caused the 1968 H3 pandemic and of the HA–esterase–fusion (HEF) glycoprotein from an influenza C virus. Structure and sequence comparisons suggest that HA subtypes may have originated by diversification of properties that affected the metastability of HAs required for their membrane fusion activities in viral infection.
GENETICS OF INFLUENZA VIRUSES Steinhauer, David A; Skehel, John J
Annual review of genetics,
01/2002, Letnik:
36, Številka:
1
Journal Article
Recenzirano
Influenza A viruses contain genomes composed of eight separate segments of
negative-sense RNA. Circulating human strains are notorious for their tendency
to accumulate mutations from one year to the ...next and cause recurrent
epidemics. However, the segmented nature of the genome also allows for the
exchange of entire genes between different viral strains. The ability to
manipulate influenza gene segments in various combinations in the laboratory
has contributed to its being one of the best characterized viruses, and studies
on influenza have provided key contributions toward the understanding of
various aspects of virology in general. However, the genetic plasticity of
influenza viruses also has serious potential implications regarding vaccine
design, pathogenicity, and the capacity for novel viruses to emerge from
natural reservoirs and cause global pandemics.
The spike glycoproteins of the lipid-enveloped orthomyxoviruses and paramyxoviruses have three functions: to recognize the receptor on the cell surface, to mediate viral fusion with the cell ...membrane, and to destroy the receptor. In influenza C virus, a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, possesses all three functions (reviewed in ref. 1). In influenza A and B, the first two activities are mediated by haemagglutinin and the third by a second glycoprotein, neuraminidase. Here we report the crystal structure of the HEF envelope glycoprotein of influenza C virus. We have identified the receptor-binding site and the receptor-destroying enzyme (9-O -acetylesterase) sites, by using receptor analogues. The receptor-binding domain is structurally similar to the sialic acid-binding domain of influenza A haemagglutinin, but binds 9-O -acetylsialic acid. The esterase domain has a structure similar to the esterase from Streptomyces scabies and a brain acetylhydrolase,. The receptor domain is inserted into a surface loop of the esterase domain and the esterase domain is inserted into a surface loop of the stem. The stem domain is similar to that of influenza A haemagglutinin, except that the triple-stranded, α-helical bundle diverges at both of its ends, and the amino terminus of HEF2, the fusion peptide, is partially exposed. The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini of HEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.
We have determined the structure of GP2 from the Ebola virus membrane fusion glycoprotein by X-ray crystallography. The molecule contains a central triple-stranded coiled coil followed by a ...disulfide-bonded loop homologous to an immunosuppressive sequence in retroviral glycoproteins, which reverses the chain direction and connects to an α helix packed antiparallel to the core helices. The structure suggests that fusion peptides near the N termini form disulfide-bonded loops at one end of the molecule and that the C-terminal membrane anchors are at the same end. In this conformation, GP2 could both bridge two membranes and facilitate their apposition to initiate membrane fusion. We also find a heptad irregularity like that in low-pH-induced influenza HA2 and a solvent ion trapped in a coiled coil like that in retroviral TMs.
Low pH induces a conformational change in the influenza virus haemagglutinin, which then mediates fusion of the viral and host cell membranes. The three-dimensional structure of a fragment of the ...haemagglutinin in this conformation reveals a major refolding of the secondary and tertiary structure of the molecule. The apolar fusion peptide moves at least 100 A to one tip of the molecule. At the other end a helical segment unfolds, a subdomain relocates reversing the chain direction, and part of the structure becomes disordered.
Abstract As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission ...between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.
The structure of a stable recombinant ectodomain of influenza hemagglutinin HA2 subunit, EHA2 (23-185), defined by proteolysis studies of the intact bacterial-expressed ectodomain, was determined to ...1.9- angstrom resolution by using x-ray crystallography. The structure reveals a domain composed of N- and C-terminal residues that form an N cap terminating both the N-terminal α -helix and the central coiled coil. The N cap is formed by a conserved sequence, and part of it is found in the neutral pH conformation of HA. The C-terminal 23 residues of the ectodomain form a 72- angstrom long nonhelical structure ordered to within 7 residues of the transmembrane anchor. The structure implies that continuous α helices are not required for membrane fusion at either the N or C termini. The difference in stability between recombinant molecules with and without the N cap sequences suggests that additional free energy for membrane fusion may become available after the formation of the central triple-stranded coiled coil and insertion of the fusion peptide into the target membrane.
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