The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice

Abstract

After receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y231H mutant, wild-type, and HA-H241Q and HA-K582I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y231H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable for the virus to remain fit. Wild-type and HA-H241Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support replication. The HA-K582I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.

Fig 1

In vitro replication kinetics of reverse genetic C58 WT and mutant viruses. MDCK (A), CCL-141 (B), DF-1 (C), A549 (D), or NHBE (E) cells were infected with reverse genetic C58 WT or mutant virus at an MOI of 0.01 PFU/cell. Supernatants were collected at the indicated time points, and virus infectious titers in MDCK cells were quantified by performing TCID₅₀ assays. Error bars represent the standard deviations of triplicate samples. Graphs are representative of two independent experiments. Statistical analysis was performed by two-way ANOVA. Asterisks indicate P values of <0.05.

Fig 2

Virulence of reverse genetic C58 WT and mutant viruses in C57BL/6J mice. Shown are the mean percent weight change (A) and survival (B) of C57BL/6J mice (n = 7) after intranasal inoculation with 1.6 × 10⁶ EID₅₀ of reverse genetic C58 WT or mutant virus. Error bars represent the standard deviations. Statistical analysis was performed by two-way ANOVA for weight loss and log-rank chi-square test for survival curves. Asterisks indicate P values of <0.05.

Fig 3

Virulence of reverse genetic C58 WT and mutant viruses in DBA/2J mice. Shown are the mean percent weight change (A) and survival (B) of DBA/2J mice (n = 12) after intranasal inoculation with 28,000 EID₅₀ of reverse genetic C58 WT and mutant viruses. Error bars represent the standard deviations. Statistical analysis was performed by two-way ANOVA for weight loss and log-rank chi-square test for survival curves. Asterisks indicate P values of <0.05.

Fig 4

Viral titers of reverse genetic C58 WT and mutant viruses in tissues of infected DBA/2J mice. Shown are the titers of reverse genetic C58 WT and mutant viruses in the nasal cavities (A), tracheas (B), lungs (C), brains (D), and kidneys (E) of infected DBA/2J mice at days 2, 4, and 7 postinfection. The ratio above each bar represents the number of mice with detectable virus titers divided by the total number of infected mice. The dotted horizontal line indicates the assay detection limit (TCID₅₀/ml = 1 log₁₀). Data from two independent experiments were combined, and the error bars represent the standard deviations. Statistical analysis was performed by one-way ANOVA. Asterisks indicate P values of <0.05.

Fig 5

Acid stability of H5N1 influenza virus. Prestandardized virus stock was diluted in PBS buffer adjusted to the indicated pH and incubated for 1 h at 37°C. The remaining infectious virus titer was quantified by performing TCID₅₀ assays with MDCK cells. Statistical analysis was performed by two-way ANOVA. Asterisks indicate P values of <0.05.