Different pH requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza A viruses

Abstract

Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH4Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents.

Figure 1

Inhibition of H5 and H3 influenza A virus replication by CQ in MDCK cells. Cells were incubated with chloroquine (CQ) after virus inoculation or mock-infection and tested for cell viability and viral RNA copies at 24 h post-infection. A) Viability of cells infected with A/Chicken/Italy/9097/97 (H5N9) and treated with increasing concentrations of CQ as detected by colorimetric test. Assays were performed as described in the text. The dotted line indicates inhibition of uninfected cell viability, the solid line indicates inhibition of infected cell viability. Results are presented as the curves that best fit the data points. B) Results of one representative experiment showing inhibition by CQ of A/Chicken/Italy/9097/97 viral RNA production. Virus infected MDCK cells were incubated for one day in the presence of 0, 5, 10, 20 or 25 μM chloroquine. Cell supernatants were used for viral RNA extraction and subjected to a quantitative real-time RT-PCR (qRRT-PCR) assay. Oseltamivir (OS; 20 nM) was used as a positive control. C and D) as in A and B), respectively, using A/Panama/2007/99-like (H3N2) virus. In D) both results obtained with inocula containing 10⁴ and 10³ TCID₅₀/ml are reported. Results in B) and D) are displayed for purely representative reasons to show that there is inhibition of virus production, and cannot be compared with each other or with those in A) and C), due to the high intra- and inter-assay variability of the qRRT PCR assay (see Ref. [29]).

Figure 2

Correlation between electric characteristics of haemagglutinin subunit 2 (HA2) and response to chloroquine of influenza A viruses. A) Correlation between EC₅₀ of chloroquine (CQ) on viral cytopathogenicity (presented as Log values, x axis) and isoelectric point of HA2 (pH value at which the protein is neutral; y axis). The line best fitting the data points is shown. Isoelectric points were calculated based on the protein sequence using the web interface in Ref. [35]. B-G) Theoretical three-dimensional models for HA2 subunits of the viruses adopted in the present study, shown in ranked order of sensitivity to chloroquine (from resistant to clinically achievable concentrations to fully sensitive). B) A/Mallard/Italy/43/01 (H7N3); C); A/Mallard/Italy/66/96 (H1N1); D) A/Chicken/Italy/9097/97 (H5N9); E) A/Turkey/Italy/220158/02 (H7N3); F) Human influenza A/Panama/2007/99-like (H3N2); G) A/New Caledonia/20/99-like (H1N1). The electrostatic potential is mapped to the protein surface as a range of colours from red (-1.8 V) to blue (+1.8 V). Equipotential surfaces are shown as dotted line grids. Protein sequence data regarding the H7N3 and H5N9 viruses can be found in GenBank with the following accession numbers: AAT37405, AAT37404, AAG60349.