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Avian Influenza A Virus polymerase can utilise human ANP32 proteins to support 1 cRNA but not vRNA synthesis 2

14 Host restriction limits the emergence of novel pandemic strains from the Influenza A 15 Virus avian reservoir. For efficient replication in mammalian cells, the avian influenza 16 RNA-dependent RNA polymerase must adapt to use human orthologues of the host 17 factor ANP32, which lack a 33 amino acid insertion relative to avian ANP32A. Here 18 we find that influenza polymerase requires ANP32 proteins to support both steps of 19 replication: cRNA and vRNA synthesis. Nevertheless, avian strains are only restricted 20 in vRNA synthesis in human cells. Therefore, avian polymerase can use human 21 ANP32 orthologues to support cRNA synthesis, without acquiring mammalian 22 adaptations. This implies a fundamental difference in the mechanism by which ANP32 23 proteins support cRNA vs vRNA synthesis.


Importance 26
In order to infect humans and cause a pandemic, avian influenza must first learn how 27 to use human versions of the proteins the virus hijacks for replication -instead of the 28 avian versions found in bird cells. One such protein is ANP32. Understanding the 29 details of how host proteins such as ANP32 support viral activity may allow the design 30 of new antiviral treatments that disrupt these interactions. In this work, we use cells 31 that lack ANP32 to unambiguously demonstrate ANP32 is needed for both steps 32 of influenza genome replication. Surprisingly however, we find that avian influenza can 33 use human ANP32 proteins for the first step of replication without any adaptation, but 34 only avian ANP32 for the second step of replication. This suggests ANP32 may have 35 replication complex bear the avian-like 627E signature. Moreover, the cRNPs 116 produced by FluPolR 627E are functional for onward replication. To conclude, our 117 study suggests that huANP32 proteins are sufficient to support avian FluPol cRNA 118 synthesis, and that host restriction acts specifically at the level of vRNA synthesis. This 119 suggests a fundamental difference in the mechanism by which the host factor ANP32 120 supports vRNA and cRNA synthesis. 121 Results 122

cRNA synthesis is inhibited in human cells lacking ANP32A/B 123
Human cells express three members of the ANP32 protein family: huANP32A, 124 huANP32B and huANP32E (23). Previous work has demonstrated that huANP32A/B 125 are functionally redundant for proviral activity, whilst huANP32E does not support 126 FluPol activity (13,14). To investigate the role of ANP32 proteins in cRNA synthesis, 127 wild type human eHAP cells (eHAP WT) and human eHAP cells in which ANP32A/B 128 have been ablated (eHAP dKO) (13) were infected with A/Puerto Rico/8/1934(H1N1) 129 (PR8) and the accumulation of segment 4 (haemagglutinin (HA)) vRNA, cRNA and 130 mRNA quantified using a tagged RT-qPCR approach (24). Following infection, there 131 was no increase in either vRNA or cRNA levels over time in the absence of ANP32A/B, 132 confirming ANP32 proteins are essential for replication ( Figure 1A,B). Significantly, no 133 increase in cRNA accumulation was observed over input, suggesting a direct role for 134 ANP32 proteins in the pioneering round of cRNA synthesis. In contrast, HA mRNA 135 transcripts increased 50-fold in dKO cells from 0 hours post infection (hpi) to 2hpi, 136 illustrating that primary transcription does not require ANP32 proteins ( Figure 1C). In 137 a separate experiment, we analysed the accumulation of segment 6 (neuraminidase 138 (NA)) vRNA, cRNA and mRNA at an early and late timepoint in eHAP WT and dKO 139 cells, using an analogous tagged RT-qPCR approach. As with segment 4, at late 140 timepoints a significant reduction in all three RNA species was observed ( Figure  141 1D,E,F). At 3hpi, a ~10 fold reduction in the accumulation of cRNA was already 142 apparent in the dKO cells, despite no difference in vRNA accumulation having yet 143 occurred. Again, this suggested a direct role for huANP32A/B in supporting cRNA 144

synthesis. 145
Human ANP32 proteins play a direct role in primary cRNA synthesis 147 To eliminate the possibility that ANP32 proteins are only required for vRNA synthesis, 148 and the lack of cRNA accumulation is an indirect effect, we made use of cRNP 149 stabilisation assays. cRNP stabilisation assays manipulate cellular conditions to allow 150 only the primary round of cRNA synthesis from incoming viral genomes (Vreede et al, 151 2004, Nilsson et al, 2017. vRNA synthesis, and consequently further secondary 152 rounds of cRNA synthesis, are inhibited. The assay involves pre-expressing FluPol 153 and NP, and then infecting cells with virus. Importantly, the FluPol that is exogenously 154 expressed is a catalytically dead mutant (PB1 D446Y) that will allow stabilisation of 155 nascent cRNA into catalytically inactive cRNPs. Cells are then drug treated with either 156 actinomycin D (ActD) or cycloheximide (CHX) during infection to inhibit the synthesis 157 of nascent viral proteins which would otherwise enable normal replication. 158 159 Initially, ActD-treated cRNP stabilisation assays were performed in eHAP WT and dKO 160 cells. ActD inhibits viral transcription, therefore in these assays only primary cRNA 161 synthesis occurs (Figure 2A). When analysed by RT-qPCR ( Figure 2B,C,D) a highly 162 significant reduction in cRNA was observed in dKO cells as compared to WT cells. As 163 expected, no accumulation of NA vRNA or mRNA occurred over background levels 164 (controls lacking transfected PB1, dotted line), in either cell type. Equal transfection 165 efficiency was confirmed by western blot ( Figure S1A). 166 167 We next established in situ assays for directly visualising IAV replication products to 168 allow single cell, spatial information to be collected alongside bulk assay readouts. To 169 achieve this, the RNA FISH assay RNAscope® was used (25). Probes were designed 170 to target either segment 6 negative sense RNA (NA vRNA probe) or positive sense RNA (NA +RNA probe). The NA +RNA probe is unable to distinguish NA cRNA/mRNA 172 due to the minimal sequence differences between these two RNA species. To counter 173 this, simultaneous cRNP stabilisation and replication assays were performed with 174 ActD treatment, to inhibit viral transcription and allow +RNA staining to be attributed 175 to cRNA. Replication assays are performed as for cRNP stabilisation assays, however 176 active polymerase is pre-expressed, so that multiple rounds of cRNA and vRNA 177 synthesis take place. Validation of the approach is shown in Figure S1B-F. In an in 178 situ cRNP stabilisation assay, +RNA accumulation could be clearly detected 3hpi in 179 the nuclei of infected WT cells ( Figure 2E). In contrast, whilst incoming vRNPs could 180 be observed in the nuclei of dKO cells, no +RNA staining was observed. This 181 corroborates the view that huANP32A/B are required for primary cRNA synthesis.  Figure 2C), a significant decrease in cRNA accumulation was 193 seen in both dKO and tKO cells compared to WT by 5hpi ( Figure 2H). A small but 194 significant difference (p=0.0018) was observed between cRNA accumulation in the 195 dKO and tKO cells, implying huANP32E may support a low level of cRNP stabilisation.
In contrast, no significant difference was observed in mRNA accumulation in the 197 presence or absence of any ANP32 proteins from 0 to 5hpi ( Figure 2I). This confirms 198 that primary transcription does not require ANP32 proteins. Comparable transfection 199 efficiency was confirmed via western blot ( Figure S2G). First, we confirmed that 5092 polymerase is also dependent on ANP32 proteins for 213 cRNA synthesis both during authentic infection ( Figure S2A,B,C) and in cRNP 214 stabilisation assays ( Figure S2D,E,F). Next, we undertook simultaneous cRNP 215 stabilisation and replication assays in human cells, where only the shorter 216 huANP32A/B/E proteins, that are incompatible with FluPol 627E, are available. We 217 pre-expressed NP with either catalytically dead 5092 FluPol 627E or 627K (cRNP 218 stabilisation assay) or WT 5092 FluPol 627E or 627K (replication assay), followed by 219 infection with either 5092K or 5092E virus in the presence of ActD. Significantly, these 220 assays allow the effect of PB2 residue 627 in either FluPolR or FluPolE to be differentiated. The resulting FluPol combinations for both cRNA and vRNA synthesis 222 are outlined in Figure 3A.  (Figure 3Aiii,iv,B,C). It has previously been described that host 239 restricted FluPol is impaired in nuclear import, due to incompatibilities with the 240 importin-alpha isoforms present in human cells (28). However, this cannot explain the 241 data obtained here, as FluPol 627E was fully functional in supporting the pioneering 242 round of cRNA synthesis and stabilisation within the nucleus during cRNP stabilisation 243 assays. As replication assays allow multiple cycles of replication, the reduced cRNA 244 synthesis observed with pre-expressed FluPol 627E is likely due to the secondary 245 effect of reduced vRNP template. Exogenous expression of chANP32A was able to 246 rescue replication for all FluPol combinations ( Figure 3D,E). 247

Discussion 248
Here we have used cell lines that lack expression of ANP32 proteins to unambiguously 249 confirm huANP32 is required to support cRNA synthesis. Nevertheless, we did not 250 observe restriction of avian virus cRNA synthesis in human cells, confirming that host 251 range restriction imparted by species differences in ANP32 acts at the level of vRNA 252 synthesis. The ANP32-stabilised replication complex is thought to be required to load an 266 encapsidating FluPol onto the promoter of the extruded strand of nascent c/vRNA. 267 Moreover, it has been proposed that the unstructured ANP32 LCAR domain may act 268 as a molecular whip, which recruits NP for loading onto the nascent RNA (11,29). 269 Previous studies have suggested that host restricted avian FluPol can generate cRNA 270 in human cells, but that the resulting cRNPs are non-functional for onwards replication 271 functional for vRNA synthesis, provided the encapsidating FluPol bears 627K for 273 onward vRNA synthesis. Therefore, functional cRNPs are produced in the absence of 274 a compatible 627-LCAR interaction, suggesting host restriction does not act at the 275 level of cRNA encapsidation. In combination, our data therefore suggests that the 627-276 LCAR interaction is required for a further mechanistic role that is specific to vRNA 277 synthesis. For example, a compatible 627-LCAR interaction could be of critical 278 importance to recruit the trans-activating FluPol (York et al., 2013). 279

280
In summary, this work has established that huANP32 is required for cRNA synthesis, 281 as well as vRNA synthesis, in human cells. Nonetheless, host restriction of avian 282 FluPol 627E acts specifically at the level of vRNA synthesis, as huANP32 paralogues 283 are sufficient for supporting avian cRNA synthesis and encapsidation. This suggests 284 a fundamental difference in the mechanism by which ANP32 proteins support cRNA 285 vs vRNA synthesis. We have also established an RNA FISH assay which allows 286 visualisation of FluPol replication products. This allows single-cell, spatial information 287 to be collated, which in future could be used to improve our understanding of the 288 spatial elements of FluPol regulation. 289

Materials and methods 290
Cells and cell culture 291