Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell’s metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-D-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme’s function from transcription to replication. Author Summary Upon infection the influenza A virus alters the metabolism of infected cells. Among others, this includes a pronounced increase in glucose metabolism. We aimed to get a better understanding of these metabolic virus-host interactions and to unravel the mechanism by which glycolytic inhibition impairs the viral life cycle. On the one hand, we observed a virus-induced upregulation of many glycolysis metabolites which could often be reversed by the administration of a glycolysis inhibitor. On the other hand, our data suggested that the inhibitor treatment severely impaired viral propagation by interfering with the regulation of the viral polymerase. This manifested in an extended phase of transcription, while replication was strongly reduced. Additionally, we assessed the safety and tolerability of the used drug in immortalized and primary cells. Our study sheds more light on metabolic virus-host interactions and provides a better understanding of metabolic interference as a potential host-targeted antiviral approach, which does not bear the risk of creating resistances.

and provides a better understanding of metabolic interference as a potential host-targeted 53 antiviral approach, which does not bear the risk of creating resistances.

Introduction
Next, we assessed the reversibility as well as metabolic and potential cytotoxic effects of the 111 2-DG treatment on cells. Here, it could be demonstrated that the strong antiviral effect of a 112 24 h treatment was quickly abolished once the inhibitor was removed (Fig 1B). The massive 113 increase of viral titers after the replacement of 2-DG with inhibitor-free medium suggested the 114 full reversibility of 2-DG-induced effects and indicated that there was no permanent cell 115 damage which is also substantiated by the literature [11]. By performing lactate 116 dehydrogenase (LDH) assays we detected no cytotoxicity within the range of used 2-DG 117 concentrations (Fig 1C), as previously demonstrated in various cell lines including A549 [26, 118 27]. Moreover, we could even observe a beneficial effect of the 2-DG treatment for the survival 119 of infected cells. With increasing 2-DG concentrations the total percentage of dead cells 120 decreased significantly 24 hours post infection (hpi) (Fig S1B). However, the results of the 121 LDH assays in combination with data obtained from trypan blue exclusions suggested a certain 122 cytostatic effect, since even though the viability of all samples was not affected, total cell counts 123 decreased with rising 2-DG concentrations (Fig S1C+D). In line with these results, a cytostatic 124 effect of 2-DG has also been observed previously in other cells [27][28][29]. Furthermore, we 125 investigated the effect of 2-DG on the metabolism in real-time via a Seahorse Analyzer. We 126 observed a very rapid and significant reduction of the glycolytic proton efflux rate (glycoPER), 127 which constitutes a direct read-out of the glycolytic rate (Fig 1D). Since a major factor to 128 calculate the glycoPER is the extracellular acidification rate (ECAR), this decreased in a similar 129 pattern as the glycoPER (Fig 1E). Simultaneously, the oxygen consumption rate (OCR) of 130 2-DG-treated cells increased quickly after the beginning of the treatment (Fig 1F). These data 131 proved the partial inhibition of glycolysis by 2-DG in a dose-dependent manner and indicated 132 that cells were able to cope with the treatment by compensating the loss of glycolytic activity 133 by upregulating cellular respiration to generate energy.

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In addition to evaluating the cytotoxicity of 2-DG, we also tested potential effects of 2-DG on 135 the innate immune response and the cellular responsiveness to viral infections. For that 136 purpose, we measured expression levels of the proinflammatory genes interleukin-6 (IL-6) and 137 C-X-C motif chemokine ligand 8 (CXCL8, protein: IL-8) as well as the interferon-stimulated genes (ISGs) DExD/H-box helicase 58 (DDX58, protein: retinoic acid inducible gene I) and 139 myxovirus resistance gene A (MxA) after stimulation with either cellular or viral RNA in the 140 presence or absence of 2-DG (Fig S1E-H). We observed a mild to more pronounced induction 141 of IL-6 ( Fig S1E) and CXCL8 (Fig S1F) with increasing concentrations of 2-DG. This finding 142 was consistent with a previous publication, reporting that nutrient shortage (also induced by 143 2-DG) triggers a cell response which resembles wound healing processes in cancer cells as 144 well as in primary cells [26]. Moreover, the mild induction of proinflammatory cytokines in the 145 presence of 2-DG might be attributed to the fact that the inhibitor can also impair glycosylation.

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This in turn gives rise to endoplasmic reticulum (ER) stress, elicited by deficient glycoproteins, 147 consequently leading to the unfolded protein response (UPR) [23] which has been 148 demonstrated to drive the production of proinflammatory cytokines [30]. On the other hand, we 149 measured no clear differences in the expression of DDX58 (Fig S1G) and MxA (Fig S1H) in 150 the presence of lower 2-DG concentrations but a moderate and significant reduction of both 151 ISGs at 25 mM of the inhibitor, when stimulated with viral RNA. Nevertheless, our data 152 confirmed that the cells were well responsive to viral stimuli, regardless of the concentration of 153 2-DG that was applied.

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Apart from the permanent cell line A549, key experiments were repeated in primary human 155 bronchial epithelial cells (HBEpCs) and genuine human lung explants (Fig S2A-E). Since the 156 used media for primary tissue contained less glucose, lower concentrations of the inhibitor S2D+E). Even though the magnitude of the inhibitory effect on glycolysis and viral replication 167 differed slightly from the data obtained with A549 cells -most likely due to distinct cellular 168 metabolic activities and lower 2-DG/glucose ratios (HBEpC) -these data suggested the save 169 use and antiviral activity of 2-DG in primary tissue. Given the remarkable impairment of IAV replication by 2-DG, we now aimed to identify the spot 173 of interference of the drug within the viral life cycle. Therefore, we checked potential changes 174 in the accumulation of various IAV proteins 24 hpi (Fig 2A) and after a single replication cycle 175 of 8 h (Fig 2B). In accordance with the strongly reduced viral titers there was also a 176 pronounced reduction of viral protein accumulation after 24 h. However, within a single 177 replication cycle we only detected rather weak differences among the accumulation of viral 178 proteins. While the accumulation of the late viral proteins polymerase acidic protein (PA) and 179 matrix protein 1 (M1) was moderately reduced, the accumulation of the early proteins NP and 180 non-structural protein 1 (NS1) was barely affected by 2-DG. Consequently, even though there 181 was a moderate reduction of some viral proteins within a single replication cycle we did not 182 consider reduced viral protein accumulation to be the main reason for the severe impact of 183 2-DG on IAV propagation.

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To rule out a general effect on the cellular protein synthesis machinery, we measured the 185 fluorescence signal of the reporter Renilla luciferase driven by a constitutive promoter in a 186 luciferase assay in the absence or presence of various concentrations of 2-DG (Fig 2C).

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Decreased signals would be an indication for an impairment of cellular transcription and/or 188 translation. Interestingly, there was no negative effect on the luciferase signal, suggesting no 189 general impairment of the cellular protein synthesis. Quite the opposite was the case when 190 high concentrations of 2-DG were used which even led to an increase of the luciferase signal.   (Fig S2F) and unaffected 8 hpi (Fig S2G), vRNA was decreased at both time 216 points (Fig S2H+I). The difference in mRNA accumulation 8 hpi might be due to a milder 2-DG 217 treatment or could be a cell-specific effect. Nevertheless, the strong reduction in vRNA 218 accumulation, limiting viral propagation, seemed to be tissue-independent.

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With this phenotype at hand, we wanted to exclude a virus strain-specific effect and additionally 220 analyzed the influence of 2-DG on viral growth, transcription, and replication of the H3N2 strain 221 A/Panama/2007/1999 (Pan/99). As for SC35M, we observed a strong dose-dependent decrease of viral titers, mRNA and vRNA 24 hpi (Fig S3A-C). Importantly, with an increase of 223 Pan/99 mRNA and a decrease of vRNA in a single cycle experiment ( Fig S3D+E) the results 224 resembled those obtained with SC35M. Therefore, glycolytic interference on IAV appears to 225 be a general phenomenon and not a virus strain-specific effect.

226
Summing up the obtained insights, the qPCR data suggested that the main cause for the 227 impairment of IAV reproduction and spread by 2-DG is the interference of the inhibitor with the 228 production of viral genome copies. Hereafter, we were especially interested in why glycolytic    (Fig 3C). In accordance with our 240 previous data on vRNA accumulation at 8 hpi (Fig 3D), the kinetic revealed that vRNA 241 accumulated at a clearly reduced rate when 2-DG was applied throughout the whole 242 experiment (Fig 4B, gray line). To verify our results, we performed strand-specific real-time 243 qPCR according to the protocol established by Kawakami et al. [33] for segment 5 (NP) and 6 244 (NA). Additionally, we analyzed segment 1 (PB2), which is the longest of the IAV gene 245 segments, to rule out effects which might be caused by the length of different segments. We  which were converted by the transfected and nascent viral proteins. Thus, we were able to 274 analyze the effect of 2-DG on the transcriptional capacity of the viral polymerase (Fig 5A) or a 275 potential effect on the replicational capacity of the polymerase since vRNA first had to be 276 synthesized from the cRNA-like template (Fig 5B). We observed that transcription was 277 significantly reduced in the presence of 2-DG (Fig 5A) which confirms the previously seen 2-DG-induced lower accumulation rate of viral mRNA in the earlier phase of the 8 h kinetic 279 (Fig 4A). On the other hand, there was no significant difference of the luciferase signal 280 between the various samples when the cRNA plasmid was transfected (Fig 5B). Even though 281 the interpretation here is less straightforward, since the transcription of vRNA in mRNA was 282 also included in this process, this suggested no reduction of the replicational capacity of the 283 viral polymerase by 2-DG.

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Additionally, we examined whether the 2-DG treatment potentially affected the durability (e.g.,

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The data presented in the last two chapters suggested that 2-DG mainly impaired IAV

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Moreover, we were interested in metabolic changes aside from glycolysis. Via hydrophilic 308 interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS), as 309 described previously [36], we analyzed major alterations of the metabolic profile of A549 cells, 310 induced by IAV infection and/or the treatment with 2-DG after 24 h (Fig 6).

311
In accordance with the literature [7,8,10], the levels of glucose and most detected glycolysis 312 intermediates were increased in infected cells, pointing towards an increase of the uptake of 313 glucose and the rate of glycolytic activity. When 2-DG was applied, several glycolytic 314 intermediates (e.g., glucose-6-phosphate (G-6-P) or F-6-P) were detected at decreased

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The effect of an IAV infection and especially of 2-DG on many nucleobase-related metabolites 357 (e.g., nucleobases, nucleosides and coenzymes with related structures) was rather mild.

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Among miscellaneous metabolites we found three candidates which were distinctly affected.

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The first one is carnitine which is important for the mitochondrial shuttle of fatty acids for

386
All described measurements so far aimed to better understand IAV and 2-DG-induced 387 metabolic alterations. However, beside these effects, we also analyzed samples which were 388 additionally supplied with mannose, a C2 epimer of glucose. Since mannose can be converted into fructose-6-phosphate (F-6-P) it should be able to bypass the inhibition by 2-DG to refuel 390 glycolysis. Hence, we expected mannose to reverse some 2-DG-induced effects. Importantly, 391 we observed this reversion, sometimes even followed by an increase, for several glycolytic 392 intermediates (e.g., G-1-P, G-6-P and F-6-P) which demonstrated the antagonistic effect of  be able to substitute each other for many of their purposes inside a cell, which would also 415 explain some results of the metabolomic data (Fig 6). Indeed, the vast majority of mannose is 416 usually shunted to glycolysis to be catabolized. The remaining mannose is mainly utilized for glycosylation and since others reported that the antiviral effect of 2-DG originated from the 419 impairment of glycosylation [25,46] rather than glycolytic inhibition, we aimed to dissect the 420 interplay of these two hexoses in the context of IAV infections and the virus-restricting effects   (Fig S5B-D). The latter can be explained by the fact that MLS prevents the redirection 435 of F-6-P to N-linked glycosylation. Therefore, more glucose will be catabolized into lactate via 436 glycolysis. Besides, we observed no significant effect on the production of viral particles (Fig   437   S5E). Subsequently, we applied MLS to infected cells which were also treated with 2-DG and 438 mannose (Fig 7B). We saw the typical reduction of viral titers when 2-DG alone was applied   (Fig S5F). To finally confirm the concept of the glycolytic rate as a 446 determinant of IAV replication, we examined the effects of 2-DG, mannose, and MLS on the 447 RNA levels of IAV after a single replication cycle of 8 h. The pattern of M1 vRNA accumulation 448 (Fig 7C) strikingly resembled the pattern of viral titers (Fig 7B). The treatment with 2-DG led 449 to a highly significant reduction of vRNA which was almost completely restored to the control 450 value by supplementation of mannose. The additional administration of MLS, however, 451 decreased the vRNA value to a similar extent as 2-DG alone did. Regarding viral mRNA 452 accumulation (Fig 7D) we observed the typical slight increase after treatment with 2-DG, but 453 barely a return to the control value when mannose was added as well. This only happened 454 when also MLS was supplemented.

455
Summarizing, these data corroborated that the antiviral activity of 2-DG mainly derived from a 456 strong impairment of the synthesis of viral genomic RNA by reducing the glycolytic rate of 457 infected host cells. Moreover, by directly or indirectly inhibiting or fueling glycolysis we found 458 a way to turn viral reproduction on and off to a certain degree.

461
Understanding the diverse interplay between the host cell metabolism and viral intruders is of 462 importance since it may create potential new strategies to counteract viral infections. In our 463 study we were able to improve our comprehension of metabolic virus-host interactions as well 464 as the mode of action of glycolytic interference on the life cycle of IAV. We observed profound 465 changes of the whole metabolic profile of infected cells (Fig 6), including especially

472
During our search for the point of interference within the viral life cycle we deduced that viral 473 protein synthesis played a subordinate role, because protein accumulation was rather mildly affected within one replication cycle (Fig 2B). Interestingly, we did not at all observe a decrease 475 in cellular protein accumulation after a 2-DG treatment, shown by steady signals of the cellular 476 control protein in our western blots and via Renilla luciferase reporter assay (Fig 2). This may 477 be indicative of a more selective effect which rather applies to viral than cellular protein 478 translation. According to the data of Fig 4 and 5 we assume that the predominant mechanism 479 which is responsible for the strong reduction of IAV multiplication is a 2-DG-mediated 480 interference with the dynamic regulation that switches the viral polymerase from a 481 transcriptase to a replicase. Even though one hypothesis proposed that viral transcription and 482 replication are stochastic without a switch mechanism [35] many studies suggest the opposite.

483
The switch process of the polymerase is still not fully understood and is probably a polymerase activity and the here presented data, it is quite possible that metabolic interference 499 via 2-DG impairs the IAV replication-associated function or the expression of one or several of 500 these viral or host factors. After all we know, however, it is also possible that there is no strong 501 switching mechanism controlling viral transcription or replication. Potentially the abundance of both processes is basically stochastic but can be modulated in favor of transcription or 503 replication in a time-dependent manner. Combining the insights from previous publications 504 with our data it is imaginable that the antiviral effect of 2-DG operates in several steps. One 505 scenario could be that inhibition by 2-DG leads to a primary antiviral effect by interfering with 506 the function of the initial transcription and replication complexes which could explain the 507 generalized lower levels of mRNA and vRNA until 7 hpi (Fig 4). A secondary effect could be 508 the seemingly selective impairment of the synthesis of some viral proteins. This includes at 509 least PA of the polymerase or RNP complex (Fig 2B). Consequently, a lack of nascent 510 polymerase complexes may have a stronger impact on replication than transcription since 511 replication requires a second polymerase for the binding of nascent cRNA and vRNA strands.

512
Alternatively or additionally, treatment with 2-DG might impair the synthesis of any of the 513 afore-mentioned modulators of the viral polymerase which may contribute to the prolonged 514 phase of transcription and the clear reduction of replication. Of course, the variety of potential 515 2-DG-mediated influences on viral replication is huge and on top of that we cannot fully exclude 516 an off-target interaction which may play a role here. However, the latter seems highly unlikely 517 based on the data we generated through the supplementation of mannose and MLS in the 518 presence of 2-DG (Fig 7). It will be interesting to examine if and how severely 2-DG influences 519 the expression or interactions of the afore-mentioned viral and cellular factors with the complex 520 replication machinery of IAV.

526
Our data indicate that the positive effect of mannose on IAV replication mainly (but not 527 necessarily exclusively) derives from fueling glycolysis via its conversion into F-6-P by MPI.

528
The partial recovery of the ATP/AMP ratio back to the physiological level in the presence of 529 2-DG and mannose (Fig 6) supports this theory. Moreover, the partial restoration of viral titers by the supplementation of pyruvate after inhibiting glycolysis substantiates the assumption that 531 glycolysis and its intermediates are crucial for virus reproduction. Probably the availability of 532 glycolytic intermediates, which are needed to fuel other pathways and to synthesize 533 macromolecules such as nucleotides and amino acids, is the most critical factor.

534
Extrapolations predicted only a very minor extra demand for energy (~1% of the total energetic 535 budget of a eukaryotic cell) to synthesize viral progeny during the characteristic time of an 536 influenza infection [77]. Therefore, we assume that a potential role of ATP in viral replication 537 may rather not be its availability for synthesis reactions.

538
As reviewed previously [78], 2-DG has various direct and indirect mechanism by which it can 539 negatively affect normal cellular functions (e.g., inhibition of glycolysis and glycosylation or 540 induction of AMPK and UPR). Therefore, a certain cytotoxicity -which heavily depends on the 541 dose and type of administration as well as the type of cell, tissue, or organism -must be 542 considered. However, we could demonstrate the tolerability and the quickness of effectivity of 543 the antimetabolite in immortalized and primary cells (Fig 1C-F, S1B, S2B+C