Time and Temperature Dependence of Influenza Virus Membrane Fusion at Neutral pH

The threat of future influenza pandemics and their potential for rapid spread, morbidity, and mortality has led to the development of pandemic vaccines. We generated seven reassortant pandemic live attenuated influenza vaccines (pLAIVs) with the hemagglutinin (HA) and neuraminidase (NA) genes derived from animal influenza viruses on the backbone of the six internal protein gene segments of the temperature sensitive, cold-adapted (ca) A/Ann Arbor/60 (H2N2) virus (AA/60 ca) of the licensed seasonal LAIV. The pLAIV viruses were moderately to highly restricted in replication in seronegative adults; we sought to determine the biological basis for this restriction. Avian influenza viruses generally replicate at higher temperatures than human influenza viruses and, although they shared the same backbone, the pLAIV viruses had a lower shutoff temperature than seasonal LAIV viruses, suggesting that the HA and NA influence the degree of temperature sensitivity. The pH of HA activation of highly pathogenic avian influenza viruses was greater than human and low-pathogenicity avian influenza viruses, as reported by others. However, pLAIV viruses had a consistently higher pH of HA activation and reduced HA thermostability compared to the corresponding wild-type parental viruses. From studies with single-gene reassortant viruses bearing one gene segment from the AA/60 ca virus in recombinant H5N1 or pH1N1 viruses, we found that the lower HA thermal stability and increased pH of HA activation were associated with the AA/60 M gene. Together, the impaired HA acid and thermal stability and temperature sensitivity likely contributed to the restricted replication of the pLAIV viruses we observed in seronegative adults. IMPORTANCEThere is increasing evidence that the HA stability of influenza viruses depends on the virus strain and host species and that HA stability can influence replication, virulence, and transmission of influenza A viruses in different species. We investigated the HA stability of pandemic live attenuated influenza vaccine (pLAIV) viruses and observed that the pLAIV viruses consistently had a less stable HA than the corresponding wild-type influenza viruses. The reduced HA stability and temperature sensitivity of the pLAIV viruses may account for their restricted replication in clinical trials.

Section: Resultsmentioning

confidence: 99%

The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus Vaccines

O’Donnell

Vogel

Matsuoka

et al. 2014

“…To investigate whether heating alone, in the absence of the low pH trigger, can also induce fusion of TBE virus with membranes, as was the case with influenza virus HA (5,11,26), we used an in vitro fusion assay based on pyrene-excimer fluorescence (6).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, influenza virus HA, which normally uses low pH as a trigger, can be induced to undergo fusion-specific structural alterations and fusion itself by heat and other destabilizing conditions (5,11,26), indicating that its energy barrier for fusion at a neutral pH is lower than the one for denaturation. Fusion at elevated temperatures has also been described for the paramyxoviruses Sendai virus and simian virus 5 (24,31).…”

Section: Vol 75 2001mentioning

confidence: 99%

“…Earlier studies with the class I fusion protein of influenza virus (hemagglutinin, HA) showed that fusion activity as well as the associated conformational changes that are normally triggered by acidic pH could also be induced by elevated temperatures and other protein-destabilizing conditions such as urea treatment (5,11,26). Since the final form is more stable, it was concluded that native HA is kinetically trapped in a metastable state (4).…”

mentioning

confidence: 99%

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Role of Metastability and Acidic pH in Membrane Fusion by Tick-Borne Encephalitis Virus

Stiasny

Allison

Mandl

et al. 2001

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.

“…In the laboratory, cleaved HAs can be induced to undergo conformational changes at neutral pH by increasing temperature, and these changes are structurally analogous to those that are triggered by acidification (45)(46)(47). Furthermore, a direct correlation is demonstrated between fusion pH and temperature at which specific mutants are triggered (i.e., high pH mutants can be triggered at lower temperature) (45).…”

Section: Importancementioning

confidence: 99%

Influenza Hemagglutinin (HA) Stem Region Mutations That Stabilize or Destabilize the Structure of Multiple HA Subtypes

Byrd-Leotis

Galloway

Agbogu

et al. 2015

Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles. IMPORTANCEThe influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components. N early every year seasonal influenza A viruses contribute to hundreds of thousands of human deaths worldwide and place an extraordinary burden on health care systems and the global economy. Of even greater concern is the unpredictable appearance of antigenically novel influenza A strains in humans, with the potential for causing devastating pandemics. A major obstacle for controlling influenza is the diversity of influenza virus A strains that circulate in the avian species, since this provides a vast genetic pool of vi...