Structural insight into the mechanism of neuraminidase inhibitor-resistant mutations in human-infecting H10N8 Influenza A virus

The emergence of drug resistance in avian influenza virus (AIV) is a serious concern for public health. Neuraminidase (NA) isolated from a fatal case of avian-origin H10N8 influenza virus infection was found to carry a drug-resistant mutation, NA-Arg292Lys (291 in N8 numbering). In order to understand the full potential of H10N8 drug resistance, the virus was first passaged in the presence of the most commonly used neuraminidase inhibitors (NAIs), oseltamivir and zanamivir. As expected, the Arg292Lys substitution was detected after oseltamivir treatment, however a novel Val116Asp substitution (114 in N8 numbering) was selected by zanamivir treatment. Next generation sequencing (NGS) confirmed that the mutations arose early (after passages 1-3) and became dominant in the presence of the NAI inhibitors. Extensive crystallographic studies revealed that N8-Arg292Lys resistance results mainly from loss of interactions with the inhibitor carboxylate, while rotation of Glu276 was not impaired as observed in the N9-Arg292Lys, a group 2 NA structure. In the case of Val116Asp, the binding mode between oseltamivir and zanamivir is different. Asp151 forms stabilized hydrogen bond to guanidine group of zanamivir, which may compensate the resistance caused by Val116Asp. By contrast, the amino group of oseltamivir is too short to maintain this hydrogen bond, which result in resistant. Moreover, the oseltamivir-zanamivir hybrid inhibitor MS-257 displays higher effectiveness to Val116Asp than oseltamivir, which support this notion. Author Summary Aside from vaccination, NAIs are currently the only alternative for the clinical treatment and prophylaxis of influenza. Understanding the mechanisms of resistance is critical to guide in drug development. In this study, two drug-resistant NA substitutions, Val116Asp and Arg292Lys, were discovered from oseltamivir and zanamivir treatment of H10N8 virus. Crystal structural analyses revealed two distinct mechanisms of these two resistant mutations and provide the explanation for the difference in susceptibility of different NAIs. Zanamivir and laninamivir were more effective against the resistant variants than oseltamivir, and Arg292Lys results in more serious oseltamivir resistance in N9 than N8 subtype. This study is well-correlated to influenza pandemic/epidemic pre-warning, as the discovery of inhibitor resistant viruses will help for new drug preparedness.


58
Four NAIs, oseltamivir, zanamivir, peramivir and laninamivir, are currently available for the 59 clinical treatment of influenza virus infections [1][2][3][4]. Oseltamivir has been extensively used due to 60 its high oral bioavailability [5], however resistance to both oseltamivir and peramivir is prevalent. 61 On the other hand, although zanamivir and laninamivir offer advantages in terms of drug 62 resistance, both inhibitors are highly polar in their active forms and therefore have lower 63 bioavailability. Furthermore, there are still many known substitutions that lead to zanamivir and 64 laninamivir resistance including Glu119Gly/Ala/Asp, Gln136Lys, Asp151Ala/Asn/Gly/Val, 65 Arg152Lys, Ile222Arg, Asp198Asn, Arg292Lys and Arg371Lys (N2 numbering) [6][7][8][9][10]. 66 Resistance to NAIs usually results from substitutions of highly conserved amino acid residues that 67 form the NA active site. The influenza NA active site contains 8 conserved catalytic residues, and 68 an additional frame of 11 residues that provides structural support to the active site [11,12]. 69 Substitution of non/semi-conserved residues has also been shown to lead to NAI resistance [13]. 70 Semi-conserved influenza NA residues such as Ile117 and Lys150 have been observed to confer 71 NAI resistance in N1 subtype NA [14,15], while Gln136 has been observed in both N1 [16] and 72 N2 [17] NA subtypes. However, the mechanisms of drug resistance related to semi-conserved 73 residues are poorly understood [6,18,19]. 74 In recent years, some new AIVs, either highly pathogenic (HPAIV) or low pathogen AIV mutation was discovered in the trachea aspirate of a 73-year-old patient who died 3 days following 80 oseltamivir treatment [27]. Therefore, H10N8 and other avian-origin influenza A viruses possess 81 a high potential for human pathogenicity and hence pose public health concern. 82 There are two phylogenetic groups of influenza A NAs (N1, N4, N5 and N8 belong to group 1, 83 while N2, N3, N6, N7 and N9 belong to group 2) [28]. Arg292Lys has been widely reported in 84 group 2 NAs (N2 and N9), while the report of Arg292Lys in a group 1 NAs has never been 85 observed in nature. Structural analysis of N9-Arg292Lys has been shown to result in unfavorable 86 Glu276 conformation for oseltamivir binding [10]. However, the mechanism of Arg292Lys 87 resistance in a group 1 N8 is not well understood due to lack of crystal structures (apo and holo). 88 In this study, we explored the potential drug-resistant substitutions of A/Jiangxi- framework or drug binding and therefore the mechanism underlying Val116Asp resistance is 94 perplexing. We have determined the binding capability (K m ) of N8 wildtype and two substitutions. 95 The data indicated that Val116Asp and Arg292Lys mutations reduce the affinity between the 96 enzyme and substrate, which results from the conformational shift of the key residues in the active 97 site. Moreover, the inhibition assay demonstrated that the oseltamivir resistance (1,000-fold) 98 caused by Arg292Lys in N8 is much less severe than that of H7N9 (100,000-fold). We have also 99 determined the apo crystal structures of N8 wild type and the two mutants and the holo structures 100 in complex with oseltamivir, zanamivir, peramivir and laninamivir. These crystal structures reveal 7 101 the underlying mechanism by which Arg292Lys and Val116Asp mutations develop resistance 102 towards NAIs.

104
In vitro NAI Selective Pressure 105 H10N8 virus was subjected to 2-fold increases in the concentration of oseltamivir (80-10240 μM) 106 and zanamivir (20-2560 μM) over 8 passages. Prior quantification by plaque assays showed that 107 the virus was resistant to oseltamivir at 13.3 μM and zanamivir at 1.33 μM. After each passage, 108 the hemagglutinin (HA) titer was determined, recorded in triplicate and used to estimate the 109 concentration of virus to be seeded in the next passage. A negative HA titer was observed in the 110 last passage indicating the absence of virus (S1 Table). to be at 28 % in the non-drug treatment control. However, just after 1 passage under oseltamivir 120 treatment, it increased to 92% and levelled off at 92% throughout the next 6 oseltamivir treatment 121 passages (Fig 1A and 2). For zanamivir treatment, the pre-exist frequency of Val116Asp was found 8 122 to be at 2%; after 1 passage, it increased quickly to 34%, and became absolute dominant (90%) 123 (Fig 1B and 2). 124 In addition to these two NA substitutions, several substitutions occurred on the HA and other 125 certain internal genes. Two HA substitutions, Lys167Glu and Ile413Thr, were pre-existing at the 126 frequency of 49% and 48% respectively, and both became dominant under oseltamivir/ zanamivir 127 treatment and kept steady along cell passages with the increase of drug concentration. Importantly,  V116Asp and 292Arg. These results suggest that synergy might exist along the dynamic changes 146 of these substitutions, which will be further studied in the future. laninamivir is similar to that of zanamivir, with 10-fold and 90-fold lower potencies, respectively.

158
On the other hand, Arg292Lys showed much higher resistance to peramivir with a 3,169-fold 159 increase in mean IC 50 value, while Val116Asp exhibited a moderate 20-fold reduced sensitivity 160 (Table 1). Interestingly, the oseltamivir-zanamivir hybrid inhibitor, MS-257 was found to be the  The crystal structures of native N8, Val116Asp and Arg292Lys were determined at resolutions of 166 1.9 Å, 1.9 Å and 1.6 Å, respectively (S2 Table). The apo structures of N8 wildtype, Val116Asp 10 167 and Arg292Lys showed similar overall active site arrangements, with some differences observed 168 regarding Gln136, Thr148, Glu276 and Tyr406 (Fig 3). Specifically, the conformations of 150-  Table). N8-Arg292Lys complexes were determined at resolutions of 1.9 180 Å, 1.8 Å, 2.0 Å and 1.8 Å for zanamivir, oseltamivir, laninamivir and peramivir, respectively (S4 181   Table). Binding of oseltamivir, zanamivir, peramivir and laninamivir to wildtype H10N8 NA 182 resembles that of typical group 1 NA binding ( substitution in N8 has no effect on the hydrogen bonds between Glu276 and Arg224, while, in N9, 203 this substitution results in loss of one hydrogen bond between Glu276 and Arg224 ( Fig 4B). These 204 differences help to explain the observation that the effect of N8-Arg292Lysoseltamivir resistance 205 is 1000-fold whereas the effect of N9-Arg292Lys resistance is 100,000-fold [10]. 206 Notably, in the structure of N8-Arg292Lys-zanamivir, the orientation of Tyr347 is the same as N8 207 wildtype, which forms hydrogen bond with the side chain of Arg371 ( Fig 4C). However, the 208 residue 347 in N9 is Aspartic acid, which side chain is not enough to form hydrogen bond with 209 Arg371 (Fig 4D). Therefore, zanamivir shows better inhibition to N8 than N9 with Arg292Lys 210 substitution (Table 1).

211
The guanidine group of zanamivir compensates the Val116Asp resistant N8 substitution 212 In order to understand the mechanism of Val116Asp resulting in more severe resistance to 213 oseltamivir than zanamivir, the zanamivir and oseltamivir complex structures of N8-Val116Asp 214 were determined at resolutions of 1.9 Å and 2.1 Å, respectively (S5 Table).  Arg292Lys and 88-fold for H7N9 N9-Arg292Lys [10], relative to the corresponding wildtype 236 NAs.

237
The Val116Lys mutation was the most intriguing N8 substitution. It is challenging to predict a 238 precise mechanism for NAI resistance, because the site of mutation is distal from the active site

248
Val116Lys also resulted in an altered conformation of the 150-loop residue Thr148 (Fig 3). It is 249 possible that the further changes observed in the loop residues 146-148 upon oseltamivir binding 250 might affect the ionic interaction of Asp151 with the oseltamivir amino group (Fig 5). Despite the 251 moderate effect on NAI inhibition, the Val116Asp substitution also resulted in a 6-fold K m increase 252 relative to the wildtype (Table 1). This indicates that Val116Asp also interferes with substrate 253 binding.
14 254 Zanamivir and laninamivir are more similar to the human sialic acid, N-acetylneuraminic acid, 255 than oseltamivir and peramivir, and therefore should be less susceptible to drug-resistance. Of the 256 4 clinical NAIs, zanamivir bound to both mutants in the most optimal conformation, whereas 257 oseltamivir binding was the least optimal. Resistance of both N8 variants to zanamivir and 258 laninamivir was also lower than that of oseltamivir and peramivir.

259
Some questions remain from the present analysis, including why peramivir was the most potent 260 inhibitor of wildtype N8 despite lacking any Tyr347 interactions. Moreover, binding of the prodrug 261 laninamivir octanoate (CS-8958) to N8 was distinct from group 1 09N1 and similar to group 2 N2, 262 which indicates that NAI binding is not always group specific.

263
In summary, our current study revealed the 4-clinical available NAI resistant substitutions for N8 264 and the underlying mechanisms have also been structurally delineated, which will help for next-