Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir

Abstract

Remdesivir (GS-5734) is a 1'-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1'-cyano modification. Compounds with modifications at the 2'-position show different patterns of inhibition. While 2'-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

Keywords: Ebola virus; GS-5734; RNA polymerase; RdRp; delayed chain termination; remdesivir; respiratory syncytial virus.

Figure 1

Chemical structures of nucleotide substrate analogues.

Figure 2

Competition between ATP and remdesivir-TP. (A) RNA substrate used in the reaction. Template and primer were both phosphorylated (p) at their 5′-ends. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. Position i allows incorporation of AMP or remdesivir-MP. (B) RNA synthesis was monitored with purified Ebola virus (EBOV) RdRp in the presence of [α-³²P]GTP, three different concentrations of the competing ATP+CTP (1 μM, 10 μM, 100 μM), and increasing concentrations of remdesivir-TP (0–1000 μM). The brackets indicate heterogeneous products containing either AMP or remdesivir-MP, or more complex mixtures due to multiple incorporation sites. Length markers (m) represent primer 4 and template 11.

Figure 3

Selective incorporation of remdesivir-MP. (A) Efficiency of nucleotide incorporation was studied with purified EBOV RdRp and RSV RdRp complexes. Template and primer were both phosphorylated (p) at their 5′-ends. RNA synthesis was monitored in the presence of [α-³²P]GTP and increasing concentrations of ATP and remdesivir-TP. G indicates incorporation of the radiolabeled nucleotide opposite template position 5. Position i allows incorporation of AMP or remdesivir-MP. Length markers (m) represent primer 4 and template 11. (B) Incorporation of AMP and remdesivir-MP by h-mtRNAP. (C) Graphic representation of data shown in (A) and (B).

Figure 4

Effect of remdesivir-MP on the efficiency of the next nucleotide incorporation event. (A) RNA synthesis was monitored with purified EBOV RdRp and RSV RdRp complexes in the presence of [α-³²P]GTP, ATP, or remdesivir-TP, respectively, and increasing concentrations of UTP. G indicates incorporation of the radiolabeled nucleotide, i indicates incorporation of AMP or remdesivir-MP, and i+1 indicates incorporation of UMP. High concentrations of UTP also promote UMP misincorporation at the following position. (B,C) Graphic representation of data shown in (A).

Figure 5

Patterns of delayed chain termination with remdesivir-TPs. (A–E, top panels) RNA synthesis was studied with recombinant EBOV RdRp on longer templates with 14 nucleotides. (A–E, bottom panels) Product formation was monitored in the presence of [α-³²P]GTP and various combinations of NTPs (100 μM) and remdesivir-TP (100 μM). In reactions containing remdesivir-TP, incorporation of i+5 is the longest significant product. The 15-nt RNA product (labeled 15) indicates minor reaction products corresponding to the length of the template plus one nucleotide that is likely generated in a template-independent manner.

Figure 6

Effective delayed chain termination of RNA synthesis by remdesivir. (A) Remdesivir-MP-dependent delayed chain termination of RNA synthesis was studied with purified EBOV RdRp. RNA synthesis was monitored in the presence of [α-³²P]GTP, CTP, and ATP or remdesivir-TP, supplemented with increasing concentrations of UTP for incorporation at position i+6, representing a 12-nt RNA product labeled 12. (B) Graphic representation of data shown in (A).

Figure 7

Inhibition of RNA synthesis with different nucleotide analogue inhibitors. RNA synthesis was monitored with EBOV RdRp and RSV RdRp in the presence of [α-³²P]-GTP and various combinations of 100 μM NTP and 100 μM NTP substrate analogues. The presence of ATP, UTP, and CTP allows full-length product formation up to position 14. The presence of UTP and CTP provides a control for mis-incorporations. Incorporated ara-AMP acts as a chain terminator, 2′-C-methyl-ATP is not incorporated, and remdesivir-MP shows delayed chain termination at position i+5. EBOV RdRp and RSV RdRp exhibit differences in patterns of RNA synthesis and its inhibition by NTP substrate analogues.