Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase

Abstract

Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3′-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses.

Remdesivir and SARS-CoV-2: Structural requirements at both nsp12 RdRp and nsp14 Exonuclease active-sites

“…However, after about 4 translocation events, we anticipate a major steric clash of the 1'-CN group with R858, suggesting that GS 441524-TP might act as a delayed chain terminator. Delayed chain termination mediated by GS 441524-MP has been demonstrated for Respiratory Syncitial virus 34 , Nipah virus 35 and Ebola virus 36 RdRps, and more relevant to this discussion for the MERS-CoV RdRp complex 36 .…”

Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV

“…Interferon beta pharmacodynamic studies. To generate a pharmacokinetic and pharmacodynamic (PK/PD) relationship for IFNb, we subcutaneously administered mouse IFNb (R + D Systems) to [18][19][20] week-old male and female Ces1c −/− or Ces1c −/− hDPP4 mice. Human equivalent dosing in mice was calculated based on the recommended dosing strategy for Betaseron (human IFNb, 0.13 million international units (MIU)/kg every other day) 54 .…”

“…Remdesivir (RDV, GS-5734) is a broad-spectrum antiviral nucleotide prodrug with potent in vitro antiviral activity against a diverse panel of RNA viruses such as Ebola virus (EBOV), Marburg, MERS-CoV, SARS-CoV, respiratory syncytial virus (RSV), Nipah virus (NiV), and Hendra virus [16][17][18] . The mechanism of RDV's anti-MERS-CoV activity is likely through premature termination of viral RNA transcription as shown in biochemical assays using recombinant EBOV, NiV, and RSV polymerases [18][19][20] . In primary human lung epithelial cell cultures, RDV is potently antiviral against circulating contemporary human CoVs, SARS-CoV (EC 50 = 0.07 µM), MERS-CoV (EC 50 = 0.07 µM), and related zoonotic bat CoVs 17,21 .…”

The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus

“…The triphosphate form of RDV (RDV-TP) was shown to inhibit the RdRp of respiratory syncytial virus (4), Nipah virus, and EBOV (7,8), which are all nonsegmented negative-sense RNA viruses. Active Ebola RdRp contains the viral L protein in complex with viral protein 35 (9,10).…”

“…Active Ebola RdRp contains the viral L protein in complex with viral protein 35 (9,10). Viral protein 35 is the functional counterpart of the P protein of respiratory syncytial virus and Nipah (7,11,12). Previously, we generated recombinant Ebola RdRp for the study of nucleotide analog inhibitors (8).…”