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Halting coronavirus polymerase

      The nucleotide analogue remdesivir is an investigational drug for the treatment of human coronavirus infection. Remdesivir is a phosphoramidate prodrug and is known to target viral RNA-dependent RNA polymerases. In this issue, Gordon et al. identify that remdesivir acts as a delayed RNA chain terminator for MERS-CoV polymerase complexes.

      Introduction

      Coronaviruses are important pathogens of many animal species, including humans. Periodic virus jumps from animals into humans have led to viral outbreaks of SARS-CoV in 2002 and MERS-CoV in 2012. The recent emergence of SARS-CoV-2 that began in 2019 has developed into a global health concern where the virus has demonstrated a strong capacity for human-to-human transmission and the ability to cross international borders. Despite the importance of these pathogenic viruses, currently there are no approved antiviral drugs for the treatment of human coronavirus infections. Although several nucleotide analogue drugs targeting viral polymerases have been approved to treat a broad range of RNA viruses, an editing exonuclease in coronaviruses provides a natural resistance to many of these small molecules. However, the drug remdesivir, originally developed for the treatment of Ebola virus (
      • Warren T.K.
      • Jordan R.
      • Lo M.K.
      • Ray A.S.
      • Mackman R.L.
      • Soloveva V.
      • Siegel D.
      • Perron M.
      • Bannister R.
      • Hui H.C.
      • Larson N.
      • Strickley R.
      • Wells J.
      • Stuthman K.S.
      • Van Tongeren S.A.
      • et al.
      Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.
      ), shows promise. Remdesivir is a phosphoramidate nucleotide analogue prodrug that is metabolized to a triphosphate form in cells and has been identified as a broad inhibitor of RNA viruses, including filo-, pneumo-, paramxyo-, and coronaviruses (
      • Lo M.K.
      • Jordan R.
      • Arvey A.
      • Sudhamsu J.
      • Shrivastava-Ranjan P.
      • Hotard A.L.
      • Flint M.
      • McMullan L.K.
      • Siegel D.
      • Clarke M.O.
      • Mackman R.L.
      • Hui H.C.
      • Perron M.
      • Ray A.S.
      • Cihlar T.
      • et al.
      GS-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses.
      ,
      • Sheahan T.P.
      • Sims A.C.
      • Graham R.L.
      • Menachery V.D.
      • Gralinski L.E.
      • Case J.B.
      • Leist S.R.
      • Pyrc K.
      • Feng J.Y.
      • Trantcheva I.
      • Bannister R.
      • Park Y.
      • Babusis D.
      • Clarke M.O.
      • Mackman R.L.
      • et al.
      Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.
      ). Remdesivir has shown effectiveness against a number of coronaviruses, with IC50 values of <0.1 μm in human airway epithelial cell models of coronavirus infection. The nucleotide analogue also prevents pathology when given prophylactically and reduces pathology when given therapeutically in animal models of coronavirus infection (
      • Sheahan T.P.
      • Sims A.C.
      • Graham R.L.
      • Menachery V.D.
      • Gralinski L.E.
      • Case J.B.
      • Leist S.R.
      • Pyrc K.
      • Feng J.Y.
      • Trantcheva I.
      • Bannister R.
      • Park Y.
      • Babusis D.
      • Clarke M.O.
      • Mackman R.L.
      • et al.
      Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.
      ). Remdesivir is currently being trialed as an antiviral therapy to treat SARS-CoV-2 infection. In this issue, Gordon et al. characterize the mechanism of remdesivir acting against the MERS-CoV polymerase complex (
      • Gordon C.J.
      • Tchesnokov E.P.
      • Feng J.Y.
      • Porter D.P.
      • Gotte M.
      The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.
      ).
      Upon infecting a host cell, the coronavirus positive-sense RNA genome is translated to produce viral polyproteins. These polyproteins are cleaved by viral proteases to yield 16 nonstructural proteins (nsp)
      The abbreviations used are: nsp
      nonstructural protein(s)
      RSV
      respiratory syncytial virus.
      responsible for replication and transcription of the viral genome. These nsp form a multisubunit complex containing many enzymatic activities, including an RNA-dependent polymerase, nsp12 (
      • Posthuma C.C.
      • Te Velthuis A.J.W.
      • Snijder E.J.
      Nidovirus RNA polymerases: complex enzymes handling exceptional RNA genomes.
      ). RNA-dependent polymerases are common features of RNA viruses, as the host cell lacks the machinery for the virus to copy its RNA genome. Being distinct from host protein machinery, these viral RNA-dependent polymerases are excellent targets for antiviral drugs.
      The RNA-dependent polymerase accepts nucleotides as substrates, and many nucleotide analogues have found utility in broadly inhibiting viral RNA synthesis (
      • Jordan P.C.
      • Stevens S.K.
      • Deval J.
      Nucleosides for the treatment of respiratory RNA virus infections.
      ). However, in addition to the nsp12 RNA polymerase, coronaviruses also encode an exonuclease, nsp14, responsible for editing mismatches that occur during viral replication, which also removes many incorporated nucleotide analogues (
      • Smith E.C.
      • Blanc H.
      • Surdel M.C.
      • Vignuzzi M.
      • Denison M.R.
      Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.
      ). This editing activity makes coronaviruses naturally resistant to several broad-spectrum RNA virus antivirals. One nucleotide inhibitor that has shown efficacy against coronaviruses in the laboratory is remdesivir, an adenosine analogue developed by Gilead Sciences (
      • Sheahan T.P.
      • Sims A.C.
      • Graham R.L.
      • Menachery V.D.
      • Gralinski L.E.
      • Case J.B.
      • Leist S.R.
      • Pyrc K.
      • Feng J.Y.
      • Trantcheva I.
      • Bannister R.
      • Park Y.
      • Babusis D.
      • Clarke M.O.
      • Mackman R.L.
      • et al.
      Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.
      ) (Gilead Sciences Update on the Company's Ongoing Response to COVID-19, Gilead Sciences, Foster City, CA). Remdesivir is now undergoing phase III clinical trials for the treatment of human coronavirus infections (
      • Agostini M.L.
      • Andres E.L.
      • Sims A.C.
      • Graham R.L.
      • Sheahan T.P.
      • Lu X.
      • Smith E.C.
      • Case J.B.
      • Feng J.Y.
      • Jordan R.
      • Ray A.S.
      • Cihlar T.
      • Siegel D.
      • Mackman R.L.
      • Clarke M.O.
      • et al.
      Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.
      ). Earlier investigations on the mechanism of remdesivir action using respiratory syncytial virus (RSV) suggested that this antiviral acted as a delayed terminator of RNA chain elongation, but there was no mechanistic understanding of how remdesivir acts against coronaviruses was unknown.
      In their new work, Gordon et al. determined the mechanism of action of remdesivir against MERS-CoV (
      • Gordon C.J.
      • Tchesnokov E.P.
      • Feng J.Y.
      • Porter D.P.
      • Gotte M.
      The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.
      ). To accomplish this, the authors used an nsp5 protease-nsp7-nsp8-nsp12 co-expression strategy using the baculovirus expression system to produce a purified complex of viral nsp8 and nsp12 for their in vitro measurements of MERS-CoV polymerase activity. Their data show that remdesivir is incorporated into the growing RNA chains, where the viral polymerase surprisingly demonstrated a preference for the analogue over the natural substrate ATP. As found for RSV, remdesivir induces termination of RNA elongation in MERS-CoV polymerase complexes. However, the termination of RNA synthesis did not occur until a further three nucleotides were incorporated into the nascent RNA, leading the authors to propose a mechanism of delayed chain termination similar to that of RSV (
      • Jordan P.C.
      • Stevens S.K.
      • Deval J.
      Nucleosides for the treatment of respiratory RNA virus infections.
      ) (Fig. 1). The authors suggest the hypothesis that because chain termination occurs three nucleotides after remdesivir is incorporated, the analogue may be protected from excision by the viral nsp14 exonuclease. Determination of how nucleotide mismatches and incorporated analogues are sensed and edited by nsp14 requires direct testing and further study. Knockout of exonuclease activity from related coronaviruses increases the potency of remdesivir, suggesting that the nsp14 exonuclease activity plays some role in limiting the antiviral effect of remdesivir (
      • Agostini M.L.
      • Andres E.L.
      • Sims A.C.
      • Graham R.L.
      • Sheahan T.P.
      • Lu X.
      • Smith E.C.
      • Case J.B.
      • Feng J.Y.
      • Jordan R.
      • Ray A.S.
      • Cihlar T.
      • Siegel D.
      • Mackman R.L.
      • Clarke M.O.
      • et al.
      Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.
      ).
      Figure thumbnail gr1
      Figure 1Mechanism of RNA termination by remdesivir. After incorporation of remdesivir by the coronavirus nsp12 RNA polymerase, a further three nucleotides are added to the growing RNA chain before the chain terminates due to the nucleotide analogue.
      The results of this study are consistent with the previous findings that remdesivir acts as a chain terminator of RNA virus polymerases. The finding that MERS-CoV polymerase preferentially incorporates remdesivir over ATP suggests a reason for the antiviral's high potency and the difficulty for coronaviruses to evolve resistance (
      • Agostini M.L.
      • Andres E.L.
      • Sims A.C.
      • Graham R.L.
      • Sheahan T.P.
      • Lu X.
      • Smith E.C.
      • Case J.B.
      • Feng J.Y.
      • Jordan R.
      • Ray A.S.
      • Cihlar T.
      • Siegel D.
      • Mackman R.L.
      • Clarke M.O.
      • et al.
      Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.
      ). Previous work has suggested that a minimal complex for coronavirus nsp12 polymerase activity required both nsp8 and nsp7 co-factors (
      • Subissi L.
      • Posthuma C.C.
      • Collet A.
      • Zevenhoven-Dobbe J.C.
      • Gorbalenya A.E.
      • Decroly E.
      • Snijder E.J.
      • Canard B.
      • Imbert I.
      One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.
      ). Structural work of the polymerase complex has shown that nsp12 binds nsp8 directly as well as interacting with a nsp7-nsp8 heterodimer, where contacts with nsp12 are mediated primarily by nsp7 (
      • Kirchdoerfer R.N.
      • Ward A.B.
      Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
      ). Although the authors co-expressed nsp7, the subunit did not co-purify with nsp8 and nsp12. One possible explanation for this may be that an insufficient amount of nsp8 was produced to form nsp7-nsp8 heterodimers. Moreover, the activity of the nsp8-nsp12 complexes indicates that nsp7 was not required for activity in this study, in contrast to previous work. This may be due to the short primer-template pairs used here to assess polymerase activity. As nsp7 has been identified as a processivity factor for the viral RNA synthesis complex, it would be interesting to know the effects of remdesivir as a chain terminator in the presence of this additional viral co-factor. Continued work to provide insights into the mechanisms of antiviral compounds is key to the development of therapies for viral infection. The work by Gordon et al. (
      • Gordon C.J.
      • Tchesnokov E.P.
      • Feng J.Y.
      • Porter D.P.
      • Gotte M.
      The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.
      ) provides key mechanistic insight into the action of remdesivir and makes important hypotheses as to the effectiveness of this nucleotide analogue against the difficult-to-target coronavirus RNA synthesis complex.

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      Linked Article

      • AGR2 Gene Function Requires a Unique Endoplasmic Reticulum Localization Motif
        Journal of Biological ChemistryVol. 287Issue 7
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          Background: Whether AGR2 promotes adenocarcinoma growth as a secreted or ER-localized protein is not known.Results: A unique carboxyl-terminal motif, KTEL, is required for AGR2 ER localization and function.Conclusion: Not all ER localization motifs are interchangeable.Significance: Specific ER localization signals may be required for protein function. AGR2's tumor-promoting effects are mediated from the ER and not as a secreted protein.
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