Impact of SARS-CoV-2 Spike Mutations on Its Activation by TMPRSS2 and the Alternative TMPRSS13 Protease.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urges better understanding of the functional motifs in the spike (S) protein and their tolerance to mutations. Here, we focused on the S2' motif, which, during virus entry, requires cleavage by a host cell protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2' motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant pseudoviruses bearing the spikes of Wuhan-Hu-1, its G614 mutant or the Delta and Omicron variants, we show that residue K814 (preceding the scissile R815) is dispensable for TMPRSS2 yet favored by the alternative TMPRSS13 protease. Activation by TMPRSS13 was drastically reduced when the SARS-CoV-2 S2' motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689), and also, the reverse effect was seen. This swap had no impact on recognition by TMPRSS2. In the Middle East respiratory syndrome coronavirus (MERS-CoV) spike, introducing a dibasic scissile motif was easily accepted by TMPRSS13 but less so by TMPRSS2, confirming that TMPRSS13 favors a sequence rich in K/R residues. Pseudovirus entry experiments in Calu-3 cells confirmed that the S2' mutations have minor impact on TMPRSS2. Our findings are the first to demonstrate which S2' residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS2 being more tolerant to variation than TMPRSS13. This preemptive insight will help to estimate the impact of S2' motif changes as they appear in new SARS-CoV-2 variants. IMPORTANCE Since its introduction in humans, SARS-CoV-2 is evolving with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike (S) protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning. Remarkably, though located in a strongly immunogenic region, the S2' cleavage motif has, thus far, remained highly conserved. This suggests that its sequence is critical for spike activation by airway proteases. To investigate this, we assessed how pseudovirus entry is affected by changes in the S2' motif. We demonstrate that TMPRSS2 readily accepts variations in this motif, whereas the alternative TMPRSS13 protease is more fastidious. The Wuhan-Hu-1, G614, Delta and Omicron spikes showed no difference in this regard. Being the first in its kind, our study will help to assess the impact of S2' variations as soon as they are detected during variant surveillance.

A broad influenza virus inhibitor acting via IMP dehydrogenase and in synergism with ribavirin.

To suppress serious influenza infections in persons showing insufficient protection from the vaccines, antiviral drugs are of vital importance. There is a need for novel agents with broad activity against influenza A (IAV) and B (IBV) viruses and with targets that differ from those of the current antivirals. We here report a new small molecule influenza virus inhibitor referred to as CPD A (chemical name: N-(pyridin-3-yl)thiophene-2-carboxamide). In an influenza virus minigenome assay, this non-nucleoside compound inhibited RNA synthesis of IAV and IBV with EC50 values of 2.3 µM and 2.6 µM, respectively. Robust in vitro activity was noted against a broad panel of IAV (H1N1 and H3N2) and IBV strains, with a median EC50 value of 0.20 µM, which is 185-fold below the 50% cytotoxic concentration. The action point in the viral replication cycle was located between 1 and 5 h p.i., showing a similar profile as ribavirin. Like this nucleoside analogue, CPD A was shown to cause strong depletion of the cellular GTP pool and, accordingly, its antiviral activity was antagonized when this pool was restored with exogenous guanosine. This aligns with the observed inhibition in a cell-based IMP dehydrogenase (IMPDH) assay, which seems to require metabolic activation of CPD A since no direct inhibition was seen in an enzymatic IMPDH assay. The combination of CPD A with ribavirin, another IMPDH inhibitor, proved strongly synergistic. To conclude, we established CPD A as a new inhibitor of influenza A and B virus replication and RNA synthesis, and support the potential of IMPDH inhibitors for influenza therapy with acceptable safety profile.

Exploration of the 2,3-dihydroisoindole pharmacophore for inhibition of the influenza virus PA endonuclease.

Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.

Favipiravir Does Not Inhibit Chikungunya Virus Replication in Mosquito Cells and Aedes aegypti Mosquitoes.

Favipiravir (T-705) is a broad-spectrum antiviral drug that inhibits RNA viruses after intracellular conversion into its active form, T-705 ribofuranosyl 5'-triphosphate. We previously showed that T-705 is able to significantly inhibit the replication of chikungunya virus (CHIKV), an arbovirus transmitted by Aedes mosquitoes, in mammalian cells and in mouse models. In contrast, the effect of T-705 on CHIKV infection and replication in the mosquito vector is unknown. Since the antiviral activity of T-705 has been shown to be cell line-dependent, we studied here its antiviral efficacy in Aedes-derived mosquito cells and in Aedes aegypti mosquitoes. Interestingly, T-705 was devoid of anti-CHIKV activity in mosquito cells, despite being effective against CHIKV in Vero cells. By investigating the metabolic activation profile, we showed that, unlike Vero cells, mosquito cells were not able to convert T-705 into its active form. To explore whether alternative metabolization pathways might exist in vivo, Aedes aegypti mosquitoes were infected with CHIKV and administered T-705 via an artificial blood meal. Virus titrations of whole mosquitoes showed that T-705 was not able to reduce CHIKV infection in mosquitoes. Combined, these in vitro and in vivo data indicate that T-705 lacks antiviral activity in mosquitoes due to inadequate metabolic activation in this animal species.

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways.

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.

Cell line-dependent activation and antiviral activity of T-1105, the non-fluorinated analogue of T-705 (favipiravir).

The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.