Aurintricarboxylic acid inhibits influenza virus neuraminidase

Abstract

We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein , was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibi-tor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location , and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro. This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus. IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge , mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a possible mechanism through which the species may not experience severe influenza virus infection in the wild. We show that the murine salivary inhibitor targets the outer surface of the influenza virus neuraminidase, possibly occluding entry to the enzymatic site rather than binding within the active site like commercially available neuraminidase inhibitors. This knowledge sheds light on how the natural inhibitors of particular species combat infection.

The significance of naturally occurring neuraminidase quasispecies of H5N1 avian influenza virus on resistance to oseltamivir: a point of concern

“…These findings highlight the possibility of mutations on residues other than catalytic or framework residues causing resistance to NAIs. In addition, the residue E276 is part of the hydrophobic pocket in the active site of NA needed to accommodate the pentyl side chain of OTV and this residue is highly conserved across all influenza A viruses (Hung et al, 2009). Furthermore, Tolentino-Lopez et al, (2013 showed that mutations outside the active site could affect the binding pattern of OTV and thus influence its resistance.…”

Properties of polysaccharides in several seaweeds from Atlantic Canada and their potential anti-influenza viral activities

“…Algal polysaccharides (250 g/mL) were screened for their antiviral activities against influenza A/PR/8/34 (H1N1) virus using a cytopathic effect (CPE) reduction method in the Madin Darby canine kidney (MDCK) cell line as described previously (Hung, Tseng et al 2009;Wang, Zhang et al 2011 …”

In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1) Influenza Viruses

“…2D). The I222 residue is highly conserved across all influenza A and B viruses and, together with R224, S246, and E276, forms the hydrophobic pocket of the active NA site (18). Of the 782 NA sequences from A(H5N1) viruses available on GenBank (accessed 15 January 2009), 24 (3%) had mutations at the I222 residue (I222T (15 strains), I222V (4 strains), I222L (2 strains), I222K (1 strain), I222F (1 strain), and I222M (1 strain)).…”