H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis

Abstract

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, KM values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (KI) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

Influenza a (H1n1) and the Resistance to Oseltamivir

“…While the first two are transmitted between birds and mammals, the last is consistent with a group of tick viruses. The influenza virus, the main one of the family, contains a single strand of ribonucleic acid (RNA) wrapped in a helical nucleocapsid, in which are present the hemagglutinin and neuraminidase, viral proteins essential for the virus replication (Stanford, 2009;van der Vries et al, 2011).…”

“…Since there is no proton input in the viral envelope, there is no release of viral RNA and viral proteins, and the viral replication cycle is interrupted (Flu Virus Today, 2009). The M2 channel in the viral membrane is the primary target of this class of anti-influenza agents, which confer specificity to the type A influenza virus, since the type B has a different membrane protein (Katzung, 2003;van der Vries et al, 2011). Unlike M2 channel blockers, which act by preventing the formation of new virus, the neuraminidase inhibitors act by preventing the release of viruses already formed and adhered to the host cell membrane.…”

“…3), thus interrupting the viral infection. Due to this mechanism, this class is active against all human influenza viruses (Flu Virus Today, 2009;Moscona, 2005;van der Vries et al, 2011). Currently, two drugs are available in Brazil: oseltamivir and zanamivir (ANVISA, 2012).…”

“…The most recent influenza pandemic was caused by a new strain of the influenza A (H1N1) virus, in its vast majority resistant to M2 channel blockers due to a single change in the M2-proton channel of serine to asparagine at position 31; fortunately the virus is sensitive to neuraminidase inhibitors (CDC, 2009;van der Vries et al, 2011). Considering this new strain's resistance to amantadine and rimantadine, the protocol of the World Health Organization recommends the use of oseltamivir for the treatment of the disease, instead of zanamivir, considering it is the most effective antiviral agent and due to the greater ease of use, since oseltamivir is available in capsules (Rungrotmongkol et al, 2009;Li et al, 2012).…”

“…Soon after this mutation was identified, several cases were found of the I223R substitution as a single mutation or in combination with the H275Y, a double mutant that proved to have high levels of resistance to oseltamivir (van der Vries et al, 2012).…”