Biology subjects

Kirchdoerfer, R. N.

Publications and source records attributed to Kirchdoerfer, R. N..

15 recordsLinked to original sources

bioRxiv2025

The 2'-endo conformation of arabinose-CTP and arabinose-UTP inhibit viral polymerases by inducing long pauses.

Key to supporting human health in the face of evolving viruses is the development of novel antiviral drug scaffolds with the potential for broad inhibition of viral families. Nucleoside analogs are a key class of drugs that have demonstrated potential for the inhibition of several viral species. Here, we evaluate arabinose nucleotides (ara-NTP) as inhibitors of the SARS-CoV-2 and poliovirus polymerases using biochemistry, biophysics and structural biology. Ara-NTPs compete poorly with their natural counterparts for incorporation into RNA by viral polymerases. However, upon incorporation, ara-NMPs induce long polymerase pausing in both SARS-CoV-2 and poliovirus polymerase RNA elongation. Our studies suggest that subsequent nucleotide incorporation is inhibited at the catalytic step due to the 2-endo sugar pucker of the incorporated ara-NMP.

biochemistry↗

bioRxiv2025

Adeno-Associated Virus 2 (AAV2) - induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression

Parvoviruses are single-stranded DNA viruses that have been modified to serve as vehicles for therapeutic transgene delivery in the form of recombinant Adeno-Associated Virus (rAAV2) vectors or rodent parvovirus-derived oncolytic agents. Infection with viruses of the Parvoviridae family induces a cellular DNA Damage Response (DDR) signal that supports virus replication. However, it remains unknown whether rAAV2 vectors or non-replicative AAV2 genomes induce cellular DDR signals, which might be deleterious to the cell. To determine the impact of AAV2/rAAV2 genomes on the integrity of the host chromosome, we have pulsed AAV2/rAAV2 infected cells with BrdU analogs followed by single-molecule imaging of the cellular replisomes and proteomic analysis of the host replication forks. We discovered that non-replicative AAV2/rAAV2 genomes are sufficient to induce replication stress on the host genome, leading to DDR signals in a dose-dependent manner. Moreover, infection with replication-competent AAV2 leads to enrichment of replication stress proteins, DNA repair factors and RNA processing machinery on cellular replication forks. However, neither the AAV2 Inverted Terminal Repeats (ITRs) that are retained in rAAV2s nor empty capsids are sufficient to induce host-cell replication stress. Strikingly, incoming AAV2 genomes associate with the single-stranded DNA binding protein RPA in host cells in a dose-dependent manner, progressively shortening cellular replication forks. These elevated levels of AAV2-induced cellular replication stress eventually leads to accumulation of DDR signals in the nucleus. Chemical inhibition of RPA activity and RNAi-mediated knockdown leads to de-repression of the AAV2 genome, increasing Rep 68/78 gene expression. Ectopic expression of RPA rescues AAV2-induced replication stress. Taken together, our findings suggest that depletion of cellular stores of RPA molecules by competing AAV2 genomes restrict viral gene expression and cause cellular DNA damage. AUTHOR SUMMARYAdeno-Associated Viruses 2 (AAV2) are modified to design therapeutic gene therapy vectors, but how they interact with the guardians of host DNA remains unknown. In this work, we show that AAV2 genomes compete with the host cell for the single-stranded DNA binding protein RPA, rendering the host vulnerable to replication stress leading to both suppression of the viral gene expression and induction of cellular DNA breaks. These findings provide insights into how gene therapies delivered at high doses could have genotoxic effects, underscoring the importance of engineering AAV2-based gene therapy platforms that express efficiently at lower doses.

microbiology↗

bioRxiv2024

A genus-specific nsp12 region impacts polymerase assembly in Alpha- and Gammacoronaviruses

Coronavirus relevancy for human health has surged over the past 20 years as they have a propensity for spillover into humans from animal reservoirs resulting in pandemics such as COVID-19. The diversity within the Coronavirinae subfamily and high infection frequency in animal species worldwide creates a looming threat that calls for research across all genera within the Coronavirinae subfamily. We sought to contribute to the limited structural knowledge within the Gammacoronavirus genera and determined the structure of the viral core replication-transcription complex (RTC) from Infectious Bronchitis Virus (IBV) using single-particle cryo-EM. Comparison between our IBV structure with published RTC structures from other Coronavirinae genera reveals structural differences across genera. Using in vitro biochemical assays, we characterized these differences and revealed their differing involvement in core RTC formation across different genera. Our findings highlight the value of cross-genera Coronavirinae studies, as they show genera specific features in coronavirus genome replication. A broader knowledge of coronavirus replication will better prepare us for future coronavirus spillovers.

biochemistry↗

bioRxiv2023

An alphacoronavirus polymerase structure reveals conserved co-factor functions

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 cryoelectron 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. Significance StatementCoronaviruses are important human and animal pathogens with a history of crossing over from animal reservoirs into humans leading to epidemics or pandemics. Betacoronaviruses, such as SARS-CoV and SARS-CoV-2, have been the focus of research efforts in the field of coronaviruses, leaving other genera (alpha, gamma, and delta) understudied. To broaden our understanding, we studied an alphacoronavirus polymerase complex. We solved the first structure of a non-betacoronavirus replication complex, and in doing so identified previously unknown, and conserved, aspects of polymerase cofactor interactions. Our work displays the importance of studying coronaviruses from all genera and provides important insight into coronavirus replication that can be used for antiviral drug development.

biochemistry↗

bioRxiv2022

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

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.

microbiology↗

bioRxiv2021

pH-dependent polymorphism of the structure of SARS-CoV-2 nsp7

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.

biochemistry↗

bioRxiv2021

From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/439712v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@de0663org.highwire.dtl.DTLVardef@757c30org.highwire.dtl.DTLVardef@12c4709org.highwire.dtl.DTLVardef@1ebd6d7_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryCryoEM and next generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.

immunology↗

bioRxiv2020

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

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) 1-3. Though the substrate targets of the viral nucleotidyl transferase are unknown, NiRAN active sites are highly conserved and essential for viral replication 3. 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.

biochemistry↗

bioRxiv2020

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

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TeaserWe revise Remdesivirs mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP

biophysics↗

bioRxiv2020

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

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.

pharmacology and toxicology↗

bioRxiv2019

Mapping polyclonal antibody responses in non-human primates vaccinated with HIV Env trimer subunit vaccines

Rational immunogen design aims to focus antibody responses to vulnerable sites on the primary antigens. Given the size of these antigens there is however potential for eliciting unwanted, off-target responses. Here, we used our electron microscopy polyclonal epitope mapping approach to describe the antibody specificities elicited by immunization of non-human primates with soluble HIV envelope trimers and subsequent repeated viral challenge. An increased diversity of epitopes recognized, and the approach angle by which these antibodies bound, constituted a hallmark of the humoral response in most protected animals. We also show that fusion peptide-specific antibodies are responsible for some neutralization breadth. Moreover, cryoEM analysis of a fully-protected animal revealed a high degree of clonality within a subset of putatively neutralizing antibodies, enabling a detailed molecular description of the antibody paratope. Our results provide important insights into the immune response against a vaccine candidate that entered into clinical trials earlier this year.

immunology↗

bioRxiv2019

Structure of the SARS-CoV NSP12 polymerase bound to NSP7 and NSP8 co-factors

Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (NSP) responsible for the replication and transcription of the viral genome. Here, we present the 3.1 [A] resolution structure of the SARS-CoV NSP12 polymerase bound to its essential co-factors, NSP7 and NSP8, using single particle cryo-electron microscopy. NSP12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is unexpectedly bound by two NSP8 co-factors. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into NSP12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.

biophysics↗

bioRxiv2019

Structure of the Ebola virus nucleoprotein - RNA complex

Ebola virus is an emerging virus capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome is carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein NP forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 [A] single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid. SynopsisThe 3.1 [A] single-particle cryo-electron microscopy structure of the RNA-bound, Ebola virus nucleoprotein helical filament provides molecular details of protein-protein and protein-RNA interactions.

biophysics↗

bioRxiv2018

An emergent Ebola virus nucleoprotein variant influences virion budding, oligomerization, transcription and replication

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measure transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013-2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NPs roles in protein structure, virion budding, and transcription and replication.

microbiology↗

bioRxiv2018

Receptor binding and proteolysis do not induce large conformational changes in the SARS-CoV spike

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S undergoes two proteolytic cleavages at S1/S2 and S2 sites necessary for efficient membrane fusion. Here, we present a cryo-EM analysis of the trimeric SARS-CoV S interactions with ACE2 and of the trypsin-cleaved S. Surprisingly, neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within S or expose the secondary cleavage site, S2. These observations suggest that S2 cleavage does not occur in the S prefusion conformation and that additional triggers may be required.

biochemistry↗