Shelly Gulati

Receptor binding specificity of recent human H3N2 influenza viruses.

BackgroundHuman influenza viruses are known to bind to sialic acid linked α2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection.ResultsViruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of α2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for α2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of α2-6 and α2-3 sialic acids, including human A549 and HeLa cells with high levels of α2-6 sialic acid, and CHO cells that have only α2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells.ConclusionAgglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of α2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of α2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with α2-6 receptor specificity.

Deletions of neuraminidase and resistance to oseltamivir may be a consequence of restricted receptor specificity in recent H3N2 influenza viruses

BackgroundInfluenza viruses attach to cells via sialic acid receptors. The viral neuraminidase (NA) is needed to remove sialic acids so that newly budded virions can disperse. Known mechanisms of resistance to NA inhibitors include mutations in the inhibitor binding site, or mutations in the hemagglutinin that reduce avidity for sialic acid and therefore reduce the requirement for NA activity.ResultsInfluenza H3N2 isolates A/Oklahoma/323/03 (Fujian-like), A/Oklahoma/1992/05 (California-like), and A/Oklahoma/309/06 (Wisconsin-like) lost NA activity on passage in MDCK cells due to internal deletions in the NA-coding RNA segment. The viruses grow efficiently in MDCK cells despite diminished NA activity. The full length NA enzyme activity is sensitive to oseltamivir but replication of A/Oklahoma/323/03 and A/Oklahoma/309/06 in MDCK cells was resistant to this inhibitor, indicating that NA is not essential for replication. There was no change in HA activity or sequence after the NA activity was lost but the three viruses show distinct, quite restricted patterns of receptor specificity by Glycan Array analysis. Extensive predicted secondary structure in RNA segment 6 that codes for NA suggests the deletions are generated by polymerase skipping over base-paired stem regions. In general the NA deletions were not carried into subsequent passages, and we were unable to plaque-purify virus with a deleted NA RNA segment.ConclusionH3N2 viruses from 2003 to the present have reduced requirement for NA when passaged in MDCK cells and are resistant to NA inhibitors, possibly by a novel mechanism of narrow receptor specificity such that virus particles do not self-aggregate. These viruses delete internal regions of the NA RNA during passage and are resistant to oseltamivir. However, deletions are independently generated at each passage, suggesting that virus with a full length NA RNA segment initiates the first round of infection.

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.

Glycan array analysis of influenza H1N1 binding and release

Influenza viruses initiate infection by attaching to sialic acid receptors on the surface of host cells. It has been recognized for some time that avian influenza viruses usually bind to terminal sialic acid that is linked in the α2-3 configuration to the next sugar while human viruses show preference for α2-6 linked sialic acid. With developments in synthetic chemistry and chemo-enzymatic methods of synthesizing quite complex glycans, it has become clear that the binding specificity extends beyond the sialic acid, and this has led to considerable interest in developing glycan reagents that could be used either as a diagnostic tool for particular influenza viruses, or to identify cells that are susceptible to infection by certain influenza viruses. Here we describe the use of the Consortium for Functional Glycomics Glycan Array to investigate binding specificity of influenza hemagglutinin and cleavage by neuraminidase, using seasonal and pandemic H1N1 influenza viruses as examples, and compare the results with published data using other array methods.

Glycosylation changes in the globular head of H3N2 influenza hemagglutinin modulate receptor binding without affecting virus virulence.

Since the emergence of human H3N2 influenza A viruses in the pandemic of 1968, these viruses have become established as strains of moderate severity. A decline in virulence has been accompanied by glycan accumulation on the hemagglutinin globular head, and hemagglutinin receptor binding has changed from recognition of a broad spectrum of glycan receptors to a narrower spectrum. The relationship between increased glycosylation, binding changes, and reduction in H3N2 virulence is not clear. We evaluated the effect of hemagglutinin glycosylation on receptor binding and virulence of engineered H3N2 viruses. We demonstrate that low-binding virus is as virulent as higher binding counterparts, suggesting that H3N2 infection does not require either recognition of a wide variety of, or high avidity binding to, receptors. Among the few glycans recognized with low-binding virus, there were two structures that were bound by the vast majority of H3N2 viruses isolated between 1968 and 2012. We suggest that these two structures support physiologically relevant binding of H3N2 hemagglutinin and that this physiologically relevant binding has not changed since the 1968 pandemic. Therefore binding changes did not contribute to reduced severity of seasonal H3N2 viruses. This work will help direct the search for factors enhancing influenza virulence.

Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: the hydrophobic side chain influences type A subtype selectivity.

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A series of influenza neuraminidase inhibitors with the pyrrolidinobenzoic acid scaffold containing lipophilic side chains at the C3 position have been synthesized and evaluated for influenza neuraminidase inhibitory activity. The size and geometry of the C3 side chains have been modified in order to investigate structure-activity relationships. The results indicated that size and geometry of the C3-side chain are important for selectivity of inhibition against N1 versus N2 NA, important type A influenza variants that infect man, including the highly lethal avian influenza.

REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes.

Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes (“editosomes”) are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 (H2F1 and H2F2). H2F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and H2F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and H2F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway.

Possible basis for the emergence of H1N1 viruses with pandemic potential from avian hosts

Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (α2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes.

Dynamic RNA holo-editosomes with subcomplex variants: Insights into the control of trypanosome editing

RNA editing causes massive remodeling of the mitochondrial mRNA transcriptome in trypanosomes and related kinetoplastid protozoa. This type of editing involves the specific insertion or deletion of uridylates (U) directed by small noncoding guide RNAs (gRNAs). Because U‐insertion exceeds U‐deletion by a factor of 10, editing increases the nascent mRNA size by up to 55%. In Trypanosoma brucei, the editing apparatus uses ~40 proteins and >1,200 gRNAs to create the functional open reading frame in 12 mRNAs. Thousands of sites are specifically recognized in the pre‐edited mRNAs and a myriad of partially edited transcript intermediates accumulates in mitochondria. The control of editing is poorly understood, but past work suggests that it occurs during substrate recognition, the initiation and progression of editing, and during the life‐cycle in different hosts. The growing understanding of the editing proteins offers clues about editing control. Most editing proteins reside in the “RNA‐free” RNA editing core complex (RECC) and in the accessory RNA editing substrate complex (RESC) that contains gRNA. Two accessory RNA helicases are known, including one in the RNA editing helicase 2 complex (REH2C). Both the RESC and the REH2C associate with mRNA, providing a rationale for the assembly of mRNA or its mRNPs, RESC, and the RECC enzyme. Identified variants of the canonical editing complexes further complicate the model of RNA editing. We examine specific examples of complex variants, differential effects of editing proteins on the mRNAs within and between T. brucei life stages, and possible control points in RNA holo‐editosomes.

DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes

Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans.