Triphosphates of the Two Components in DESCOVY and TRUVADA are Inhibitors of the SARS-CoV-2 Polymerase

Steffen Jockusch; Chuanjuan Tao; Xiaoxu Li; Thomas K. Anderson; Minchen Chien; Shiv Kumar; James J. Russo; Robert Kirchdoerfer; Jingyue Ju

We tested the ability of the active triphosphate forms of both tenofovir and emtricitabine to be incorporated by the SARS-CoV-2 RdRp. The RdRp of SARS-CoV-2 (referred to as nsp12) and its two protein cofactors (nsp7 and nsp8), whose homologs were shown to be required for the processive polymerase activity of nsp12 in SARS-CoV, 24,25 were cloned and purified as previously described. 11 We then performed polymerase extension assays with UTP + TFV-DP, and UTP + ATP + Ec-TP, following the addition of a pre-annealed RNA template and primer to a pre-assembled mixture of the SARS-CoV-2 RdRp (nsp12) and the two cofactor proteins (nsp7 and nsp8). The primer extension products from the reaction were analyzed by MALDI-TOF-MS. The RNA template and primer, corresponding to the 3' end of the SARS-CoV-2 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.03.022939 doi: bioRxiv preprint genome, were used for the polymerase assay; their sequences are indicated at the top of Fig. 2 . In the case of tenofovir diphosphate (TFV-DP) that contains an adenine base, because there are two A's in a row followed by a U in the next available positions of the template for RNA polymerase extension downstream of the priming site, we added both UTP and TFV-DP in the extension reaction. Based on results we previously obtained for TFV-DP, 11 we reduced the ratio of UTP and TFV-DP from 1:10 to 1:100 in this current experiment. If TFV-DP acts as a permanent terminator of the RdRp reaction, we would ideally observe a peak in the spectrum at the third primer extension position caused by incorporation of two Us and one TFV-DP. In the case of emtricitabine triphosphate (Ec-TP), which is a C analogue, we need to extend the primer to the first available G in the template sequence. Since the next available positions in the template consist of two As, four Us, another A and a G, we included UTP, ATP and Ec-TP in the extension reaction. Thus, if Ec-TP serves as a permanent inhibitor of the RdRp reaction, we would ideally observe a peak in the spectrum at the eighth primer extension position caused by incorporation of two Us, four As, another U and one Ec-TP.