Highlights d B.1.1.7, B.1.351, and P.1 do not show augmented host cell entry d Entry inhibitors under clinical evaluation block all variants d B.1.351 and P.1 can escape from therapeutic antibodies d B.1.351 and P.1 evade antibodies induced by infection and vaccination

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2’ site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. IMPORTANCE Cell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2’ cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi’s sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.

The SARS-Coronavirus-2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS-CoV-2-S) with the ACE2 receptor and activation by proteases, in particular TMPRSS2. Viruses can also spread through fusion of infected with uninfected cells. We therefore analyzed cell-cell fusion activity of SARS-CoV-2-S with regard to the requirements for ACE2 expression, proteolytic activation, and sensitivity to inhibitors and compared it to SARS-CoV-S. We compared S-protein-driven fusion with target cells recombinantly overexpressing ACE2, TMPRSS2, or both. SARS-CoV-2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while the reverse observation was made for SARS-CoV-S. TMPRSS2-mediated effects were inhibited by the serine protease inhibitor Camostat. Effector-target-cell fusion by SARS-CoV-2-S was only affected by Camostat when receptor expression was limiting or when the S1/S2 cleavage site was mutated. Mutational ablation of the SARS-CoV-2-S S2’ cleavage site abrogated any effects of TMPRSS2 on fusion. Mutation of the SARS-CoV-2-S S1/S2 cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting. When both factors were abundant, initial target-effector-cell fusion was unaltered, but syncytia remained smaller over time. Overall, its polybasic cleavage site renders SARS-CoV-2-S-mediated cell-cell fusion less dependent on TMPRSS2 activity on target cells. Unexpectedly, we observed enhancement of SARS-CoV-2-S-mediated fusion by Bromhexine, another TMPRSS2 inhibitor. This effect required intact proteolytic cleavage sites, suggesting interference of Bromhexine with proteolytic priming, but not in a therapeutically desired way. Infection with SARS-CoV-2-S-pseudotyped particles clearly differed in the requirements for proteolytic activation from cell-cell fusion. TMPRSS2 strongly enhanced infection, which was reversed by Camostat but not by Bromhexine.IMPORTANCECell-cell fusion allows the virus to infect additional target cells without the need to produce free virus. Fusion likely also contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2’ cleavage site is essential for activation by TMPRSS2 in trans and unravel important differences between SARS-CoV and SARS-CoV-2. Bromhexine, an inhibitor of the TMPRSS2 protease, is currently tested in clinical trials against COVID-19. Our results indicate that Bromhexine does not inhibit SARS-CoV-2-S-mediated particle entry and enhances fusion. We therefore caution against overly optimistic use of Bromhexine in higher dosage in clinical trials or as a therapy, at least until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similar to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on SARS-CoV-2-S-mediated fusion and may therefore currently represent a better choice in therapeutic regimens for COVID-19.

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of UK, South Africa and Brazil variant into human cells is susceptible to blockade by entry inhibitors. In contrast, entry of the South Africa and Brazil variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment and was less efficiently inhibited by serum/plasma from convalescent or BNT162b2 vaccinated individuals. These results suggest that SARS-CoV-2 may escape antibody responses, which has important implications for efforts to contain the pandemic.