These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses

These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses. IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. cells (DCs). In contrast, the HA-Y17H mutation reduced computer virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial Rabbit polyclonal to ACTG cell cultures and attenuated computer virus replication, computer virus spread, the severity of contamination, and cellular infiltration in the lungs of mice. Normalizing computer virus infection and weight loss in mice by inoculating them GSK547 with Y17H computer virus at a dose 500-fold higher than that of WT computer virus revealed that this destabilized mutant computer virus brought on the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early contamination in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies uncover that HA stability may regulate pathogenicity by modulating IFN responses. IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza computer virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guideline response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity by modulating GSK547 type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations. infectivity and replication to avoid extracellular inactivation in the respiratory tract (16). To test this hypothesis, we studied contamination with A/Tennessee/1-560/2009 (H1N1), a 2009 pandemic computer virus, and two viruses with HA stability-altering mutations (Y17H and R106K). The wild-type (WT) HA protein is activated at pH 5.5, whereas a Y17H mutation in the HA1 fusion peptide pocket increases the activation pH to 6.0 and an R106K mutation in the HA2 coiled-coil core decreases the activation pH to 5.3 (15). Neither mutation altered HA protein expression, cleavage, maturation, receptor-binding avidity, or receptor-binding specificity. Both mutant viruses exhibited replication kinetics similar to those of the WT computer virus in MDCK cells when inoculated at a multiplicity of contamination (MOI) of 0.01 PFU/cell; however, the Y17H computer virus had reduced replication and was less lethal than the WT computer virus in mice (15). The objective of this study was to use a mouse model to determine the mechanism by which HA stability regulates A/H1N1/2009 replication and pathogenicity. (This article was submitted to an online preprint archive [48].) RESULTS Y17H computer virus is usually attenuated for infectivity, replication, and GSK547 virulence in mice. The HA protein of the A/Tennessee/1-560/2009 (H1N1) WT computer virus was previously shown to be activated for membrane fusion, or in the absence of target cells inactivated by low-pH buffer, at a midpoint pH of 5.5. HA stalk mutations HA1-Y17H and HA2-R106K altered the HA stability to pH 6.0 and 5.3, respectively, yet these mutated proteins retained similar expression levels, cleavage, and receptor-binding specificities (15, 49). To investigate the mechanisms by which HA stability alters infectivity and pathogenicity, we inoculated groups of DBA/2 mice intranasally with various doses of WT, Y17H, and R106K viruses generated by reverse genetics (r.g.) approaches. The mouse 50% infectious dose (MID50) of these viruses decreased with decreasing HA activation pH (Table 1); thus, increased HA stability was associated with increased infectivity. WT computer virus had a mouse 50% lethal dose (MLD50) GSK547 value of 11,000 PFU, the R106K mutation increased the MLD50 to 20,100 GSK547 PFU, and 80% of Y17H virus-infected mice survived contamination with 375,000 PFU, the highest dose tested (Table 1). At comparative doses, the WT and R106K viruses induced comparable weight loss, whereas the Y17H computer virus caused substantially less weight loss (Fig. 1A to ?toC).C). For example, at a dose of 750 PFU, mice in the WT and R106K virus-infected groups exhibited approximately 10%.