This may explain why this variant not only shows higher , but also earlier  transmissibility in animal models. 0.05; *, P 0.05; **, P 0.01; ****, P 0.0001 (Fishers LSD test; versus 33C condition).(TIF) ppat.1009500.s005.tif (482K) GUID:?14952FFF-B4C5-499F-83C6-D5A2724204DB S2 Fig: Pseudovirus entry is similar at 33C and 37C. S-pseudotyped MLV particles were produced at their optimal production heat (33C or 37C), harvested and used for transduction of HEK293T target cells expressing the appropriate receptor and TMPRSS2. Transduction was carried out for 2 h at either 33C or 37C, after which particles were removed, new medium was added and further incubation was done at 33C. At day SU5614 3 post transduction, particle entry was measured by luminescence read-out (mean SEM from three experiments). ns, P 0.05 (two-tailed unpaired t-test, 37C versus 33C).(TIF) ppat.1009500.s006.tif (654K) GUID:?2BB9D721-11F3-4473-ABEE-B8E484EC4BF5 S3 Fig: Effect of production temperature on infectivity of S-pseudotyped VSV particles. GFP-encoding pseudoviruses bearing different S-proteins were produced in BHK-21J cells at either 32 or 37C. Next, they were transduced into target cells, i.e. Vero E6 for SARS-S- and SARS-2-S-bearing pseudoviruses; Huh-7 for MERS-S; or 16HBE for 229E-S, and incubated at SU5614 37C. One day later, the number of GFP-expressing cells was quantified by high-content imaging. N = 2, performed in triplicate. **, P 0.01; ***, P 0.001; ****, SU5614 P 0.0001 (two-tailed unpaired t-test; 37C versus 32C).(TIF) ppat.1009500.s007.tif (648K) GUID:?C0283D0D-AF6B-4BDD-B734-4F04810A80C8 S4 SU5614 Fig: Expression of coronavirus receptors in Calu-3 and Vero E6 cells, and in human lung tissue and human nasal tissue. The heatmap shows mRNA levels of the receptor transcripts (relative to -actin), determined by RT-qPCR. The Table shows the primer sequences used for RT-qPCR analysis.(TIF) ppat.1009500.s008.tif (800K) GUID:?A738C35C-7F8B-4808-AECB-4FE539A89222 S5 Fig: Exogenous trypsin restores the entry defect in Calu-3 cells of S1/S2 loop mutant SARS-2-S pseudoviruses. The pseudoparticles were allowed to bind to Calu-3 cells for 1 h at 4C, after which unbound particles were removed and DMEM with 10 g/ml TPCK-trypsin SU5614 was added. After 2 h at 37C, the medium was replaced by Calu-3 growth medium. Results are the mean SEM; N = 3. ***, P 0.001 (two-tailed unpaired t-test; trypsin-treated versus -untreated condition).(TIF) ppat.1009500.s009.tif (368K) GUID:?EF2FFF7B-3B81-4227-80B1-76F6E560DF44 S1 Data: Numerical values used to generate graphs. (XLSX) ppat.1009500.s010.xlsx (80K) GUID:?55A95DBA-3AB3-4A72-83A5-5ADBFA164D96 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract 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 human respiratory tract. First, the S proteins exhibit an intrinsic heat 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 33C instead of 37C, 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 37C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not Rabbit Polyclonal to TPH2 (phospho-Ser19) 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 37C, and, enhances its use.