An alphacoronavirus polymerase structure reveals conserved co-factor functions (preprint)

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs.

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1Omicron (preprint)

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1-7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

pH-dependent polymorphism of the structure of SARS-CoV-2 nsp7 (preprint)

The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at acidic and basic conditions, SARS-CoV-2 nsp7 has a helical bundle folding at neutral pH. Remarkably, the 4 helix shows gradual dislocation from the core 2-3 structure as pH increases from 6.5 to 7.5. The protonation state of residue H36 contributes to the change of nsp7s intramolecular interactions, and thus, to the structural variation near-neutral pH. Spin-relaxation results revealed that all three loop regions in nsp7 possess dynamic properties associated with this structural variation.

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2 (preprint)

Coronaviruses, like SARS-CoV-2, encode a nucleotidyl transferase in the N-terminal NiRAN domain of the non-structural protein (nsp) 12 protein within the RNA dependent RNA polymerase (RdRP). Though the substrate targets of the viral nucleotidyl transferase are unknown, NiRAN active sites are highly conserved and essential for viral replication. We show, for the first time, the detection and sequence location of GMP-modified amino acids in nidovirus RdRP-associated proteins using heavy isotope-assisted MS and MS/MS peptide sequencing. We identified lys-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of nucleotidylation in vitro that uses a phosphoramide bond to covalently attach with GMP. In SARS-CoV-2 replicase proteins, we demonstrate a unique O-linked GMP attachment on nsp7 ser-1, whose formation required the presence of nsp12. It is clear that additional nucleotidylation sites remain undiscovered, which includes the possibility that nsp12 itself may form a transient GMP adduct in the NiRAN active site that has eluted detection in these initial studies due to instability of the covalent attachment. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers (preprint)

Coronavirus Disease 2019 (COVID-19) results from an infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the third coronavirus outbreak to plague humanity this century. Currently, the most efficacious therapeutic against SARS-CoV-2 infection is the Remdesivir (RDV), an adenine-like ribonucleotide analogue that is very efficiently incorporated by the SARS-CoV-2 replicase. Understanding why RDV is so well incorporated will facilitate development of even more effective therapeutics. Here, we have applied a high-throughput, single-molecule, magnetic-tweezers platform to study thousands of cycles of nucleotide addition by the SARS-CoV-2 replicase in the absence and presence of RDV, a Favipiravir-related analog (T-1106), and the endogenously produced ddhCTP. Our data are consistent with two parallel catalytic pathways of the replicase: a high-fidelity catalytic (HFC) state and a low-fidelity catalytic (LFC) state, the latter allowing the slow incorporation of both cognate and non-cognate nucleotides. ddhCTP accesses HFC, T-1106 accesses LFC as a non-cognate nucleotide, while RDV efficiently accesses both LFC pathway. In contrast to previous reports, we provide unequivocal evidence against RDV functioning as a chain terminator. We show that RDV incorporation transiently stalls the replicase, only appearing as termination events when traditional, gel-based assays are used. The efficiency of ddhCTP utilization by the SARS-CoV-2 replicase suggests suppression of its synthesis during infection, inspiring new therapeutic strategies. Use of this experimental paradigm will be essential to the development of therapeutic nucleotide analogs targeting polymerases.

A Library of Nucleotide Analogues Terminate RNA Synthesis Catalyzed by Polymerases of Coronaviruses Causing SARS and COVID-19 (preprint)

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of additional nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2 or 3 modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses exonuclease activity. We examined these nucleotide analogues with regard to their ability to be incorporated by the RdRps in the polymerase reaction and then prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (Carbovir triphosphate, Ganciclovir triphosphate, Stavudine triphosphate, Entecavir triphosphate, 3-O-methyl UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2-O-methyl UTP), and 3 did not terminate the polymerase reaction (2-fluoro-dUTP, 2-amino-dUTP and Desthiobiotin-16-UTP). The coronavirus genomes encode an exonuclease that apparently requires a 2 -OH group to excise mismatched bases at the 3-terminus. In this study, all of the nucleoside triphosphate analogues we evaluated form Watson-Cricklike base pairs. All the nucleotide analogues which demonstrated termination either lack a 2-OH, have a blocked 2-OH, or show delayed termination. These nucleotides may thus have the potential to resist exonuclease activity, a property that we will investigate in the future. Furthermore, prodrugs of five of these nucleotide analogues (Brincidofovir/Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA approved for other viral infections, and their safety profile is well known. Thus, they can be evaluated rapidly as potential therapies for COVID-19.