Aurintricarboxylic Acid Is a Potent Inhibitor of Influenza A and B Virus Neuraminidases

Hashem, Anwar M.; Flaman, Anathea S.; Farnsworth, Aaron; Brown, Earl G.; Van Domselaar, Gary; He, Runtao; Li, Xuguang

Although ATA has been reported to have antiviral activity against human immunodeficiency virus [19, 20] , vesicular stomatitis virus [17] , SARS-CoV [21] and vaccinia virus [22] , the mechanism by which ATA inhibits such a diverse group of viruses has remained largely undefined. Various biological activities have been ascribed to ATA. It has been characterized as an inhibitor of nucleic acid processing enzymes [19] , nucleases [18] , kinase [40] and the JAK-STAT pathway [41] , and has been described as an anti-apoptotic factor [40, 42] and insulin-like growth factor [43] . The variety of cellular activities associated with ATA has complicated the elucidation of its antiviral mechanism. While it was previously proposed that ATA inhibits influenza viruses by compromising viral RNA-dependent polymerase [44] , we present evidence suggesting that the antiviral activities of ATA could be attributed to the inhibition of the viral neuraminidase. Specifically, we first observed aggregated viral particles on the surface of cells, reminiscent of previous reports by other investigators studying NA inhibitors or NA-defective viruses [29, 30] . This observation led us to conduct a cell-free NA assay, revealing that ATA substantially inhibited the enzymatic activities of both viral and recombinant NA. The difference in the magnitude of inhibition between H1N1 (PR8 and NC) and H3N2 (NY) viruses in infected cultures ( Fig.1 and Fig. 4) is not fully understood. Interestingly, no marked differences were observed in the neuraminidase inhibition tests, i.e. the IC 50 between the three strains (Fig. 9A) . The reasons for the discrepancies between tissue culture studies and in vitro enzymatic studies are still unclear. It is plausible that the in vitro enzymatic studies may not accurately reflect the events in live cell cultures. Moreover, the IC 50 in the oseltamivir-resistant strain (H274Y) only increased by 9-fold (Fig. 10B) . We think that ATA is still a potent inhibitor for the oseltamivir-resistant strain when compared with oseltamivir in inhibiting the H274Y mutant, for which an over 700-fold increase of IC 50 is needed [33] . It would be interesting to investigate potential ATA resistant mutants with respect to NA amino acids mutations.