Structures of the promoter-bound respiratory syncytial virus polymerase.

The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs1. The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome2. However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV.

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus.

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N-RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

Deletion of sigB Causes Increased Sensitivity to para-Aminosalicylic Acid and Sulfamethoxazole in Mycobacterium tuberculosis.

Although the de novo folate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigB gene in Mycobacterium tuberculosis was deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosis ΔsigB strain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabB was observed in the ΔsigB strain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigB deletion mutant and wild-type strain, and the results showed that sigB deletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro Furthermore, we found that deleting pabC also caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigB affects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.

Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis.

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.

Optimal Conditions for In Vitro Assembly of Respiratory Syncytial Virus Nucleocapsid-like Particles.

The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N0P). Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.

Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes.

RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (-) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5'-RNA capping and methylation, and 3' end polyadenylation. Genome replication includes (+) RNA antigenome and (-) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus.

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

Efficient purification and assembly of ribonucleoprotein complex for interaction analysis by MST assay coupled with GaMD simulations.

Here, we describe a generic protocol for monitoring protein-RNA interaction using a cleavable GFP fusion of a recombinant RNA-binding protein. We detail each expression and purification step, including high salt and heparin column for contaminant RNA removal. After the assembly of RNA into the ribonucleoprotein complex, the MicroScale Thermophoresis assay enables the binding affinity to be obtained quickly with a small amount of sample. Further Gaussian accelerated molecular dynamics simulations allow us to analyze protein:RNA interactions in detail. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020).

Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA.

The M2-1 protein of human respiratory syncytial virus (HRSV) is a transcription anti-terminator that regulates the processivity of the HRSV RNA-dependent RNA polymerase (RdRP). Here, we report a crystal structure of HRSV M2-1 bound to a short positive-sense gene-end RNA (SH7) at 2.7 Å resolution. We identified multiple critical residues of M2-1 involved in RNA interaction and examined their roles using mutagenesis and MicroScale Thermophoresis (MST) assay. We found that hydrophobic residue Phe23 is indispensable for M2-1 to recognize the base of RNA. We also captured spontaneous binding of RNA (SH7) to M2-1 in all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method. Both experiments and simulations revealed that the interactions of RNA with two separate domains of M2-1, the zinc-binding domain (ZBD) and the core domain (CD), are independent of each other. Collectively, our results provided a structural basis for RNA recognition by HRSV M2-1.

Cryo-EM structure of the respiratory syncytial virus RNA polymerase.

The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.

MgrB affects the acid stress response of Escherichia coli by modulating the expression of iraM.

Although MgrB is established to be a feedback inhibitor of the PhoP/Q system in Escherichia coli, the biological functions of MgrB remain largely unknown. To explore new functions of MgrB, a comparative transcriptome analysis was performed (E. coli K-12 W3110 ΔmgrB vs E. coli K-12 W3110). The results showed that many genes involved in acid stress are upregulated, suggesting that MgrB is related to acid sensitivity in E. coli. The survival rates under acid stress of the ΔmgrB mutant and wild-type showed that deletion of mgrB resulted in acid resistance. According to previous research, we deleted phoP, phoQ and iraM in the ΔmgrB mutant, and found that further deletion of phoP/phoQ only partially restored acid sensitivity. Additionally, we found that deletion of mgrB resulted in increased accumulation of RpoS during the exponential growth phase, which could be blocked by further deletion of iraM. Mutation of iraM or rpoS completely suppressed the effect of mgrB mutation on acid resistance. Taken together, the data suggest that MgrB affects the acid resistance of E. coli by modulating the expression of iraM, but not completely through PhoP/Q. This indicates that MgrB may have other protein interactors aside from PhoQ, which merits further investigation.

Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis.

MarR family proteins are transcriptional regulators that control expression of bacterial proteins involved in metabolism, virulence, stress responses and multi-drug resistance, mainly via ligand-mediated attenuation of DNA binding. Greater understanding of their underlying regulatory mechanism may open up new avenues for the effective treatment of bacterial infections. To gain molecular insight into the mechanism of Rv2887, a MarR family protein in M. tuberculosis, we first showed that it binds salicylate (SA) and para-aminosalicylic acid (PAS), its structural analogue and an antitubercular drug, in a 1:1 stoichiometry with high affinity. Subsequent determination and analysis of Rv2887 crystal structures in apo form, and in complex with SA, PAS and DNA showed that SA and PAS bind to Rv2887 at similar sites, and that Rv2887 interacts with DNA mainly by insertion of helix α4 into the major groove. Ligand binding triggers rotation of the wHTH domain of Rv2887 toward the dimerization domain, causing changes in protein conformation such that it can no longer bind to a 27 bp recognition sequence in the upstream region of gene Rv0560c. The structures provided here lay a foundation for the design of small molecules that target Rv2887, a potential new approach for the development of anti-mycobacterials.

Crystal structure of Rv3899c(184-410), a hypothetical protein from Mycobacterium tuberculosis.

Rv3899c is a hypothetical protein from Mycobacterium tuberculosis which is conserved across mycobacteria. It is predicted to be secreted and has been found in culture filtrates. It has been proposed as a potential vaccine candidate; however, its biological function is unknown. Here, the global structure of Rv3899c(184-410), a fragment of Rv3899c, is reported. The structure resembles the shell of a sea snail, and its N- and C-termini form two relatively independent compact domains: an α/ß/α sandwich folding domain and an α-helix bundle domain. There are no reported protein structures for any Rv3899c homologues; this structure provides the first structural glimpse of a new protein family consisting of Rv3899c and its homologues.

Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance.

The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance-associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance-associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.