Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

Single-molecule FRET for virology: 20 years of insight into protein structure and dynamics.

Although viral protein structure and replication mechanisms have been explored extensively with X-ray crystallography, cryo-electron microscopy, and population imaging studies, these methods are often not able to distinguish dynamic conformational changes in real time. Single-molecule fluorescence resonance energy transfer (smFRET) offers unique insights into interactions and states that may be missed in ensemble studies, such as nucleic acid or protein structure, and conformational transitions during folding, receptor-ligand interactions, and fusion. We discuss the application of smFRET to the study of viral protein conformational dynamics, with a particular focus on viral glycoprotein dynamics, viral helicases, proteins involved in HIV reverse transcription, and the influenza RNA polymerase. smFRET experiments have played a crucial role in deciphering conformational changes in these processes, emphasising the importance of smFRET as a tool to help elucidate the life cycle of viral pathogens and identify key anti-viral targets.

High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization.

Many viruses form highly pleomorphic particles. In influenza, virion structure is of interest not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline, a method well-suited to the study of large numbers of pleomorphic structures, to image many thousands of individual influenza virions; gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super-resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. We also showed that viral RNP complexes are located preferentially within Archetti bodies when these were observed at filament ends, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses.

Cryopreserved Kidney Epithelial (Vero) Cell Monolayers for Rapid Viral Quantification, Enabled by a Combination of Macromolecular Cryoprotectants.

Plaque assays quantify the amount of active, replicating virus to study and detect infectious diseases by application of samples to monolayers of cultured cells. Due to the time taken in thawing, propagating, plating, counting, and then conducting the assay, the process can take over a week to gather data. Here, we introduce assay-ready cryopreserved Vero monolayers in multiwell plates, which can be used directly from the freezer with no cell culture to accelerate the process of plaque determination. Standard dimethyl sulfoxide cryopreservation resulted in just 25% recovery, but addition of polyampholytes (macromolecular cryoprotectants) increased post-thaw recovery and viability in 12- and 24-well plate formats. Variability between individual wells was reduced by chemically induced ice nucleation to prevent supercooling. Cryopreserved cells were used to determine influenza viral plaques in just 24 h, matching results from nonfrozen controls. This innovation may accelerate viral detection and quantification and facilitate automation by eliminating extensive cell culturing.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Real-time analysis of single influenza virus replication complexes reveals large promoter-dependent differences in initiation dynamics.

The viral RNA (vRNA) genome of influenza viruses is replicated by the RNA-dependent RNA polymerase (RNAP) via a complementary RNA (cRNA) intermediate. The vRNA promoter can adopt multiple conformations when bound by the RNAP. However, the dynamics, determinants, and biological role of these conformations are unknown; further, little is known about cRNA promoter conformations. To probe the RNA conformations adopted during initial replication, we monitored single, surface-immobilized vRNA and cRNA initiation complexes in real-time. Our results show that, while the 3' terminus of the vRNA promoter exists in dynamic equilibrium between pre-initiation and initiation conformations, the cRNA promoter exhibited very limited dynamics. Two residues in the proximal 3' region of the cRNA promoter (residues absent in the vRNA promoter) allowed the cRNA template strand to reach further into the active site, limiting promoter dynamics. Our results highlight promoter-dependent differences in influenza initiation mechanisms, and advance our understanding of virus replication.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter.

The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction.

Engineering stress as a motivation for filamentous virus morphology.

Many viruses are pleomorphic in shape and size, with pleomorphism often thought to correlate with infectivity, pathogenicity, or virus survival. For example, influenza and respiratory syncytial virus particles range in size from small spherical virions to filaments reaching many micrometers in length. We have used a pressure vessel model to investigate how the length and width of spherical and filamentous virions can vary for a given critical stress and fluorescence super-resolution microscopy along with image analysis tools to fit imaged influenza viruses to the model. We have shown that influenza virion dimensions fit within the theoretical limits of the model, suggesting that filament formation may be a way to increase an individual virus's volume without particle rupture. We have also used cryoelectron microscopy to investigate influenza and respiratory syncytial virus dimensions at the extrema of the model and used the pressure vessel model to explain the lack of alternative virus particle geometries. Our approach offers insight into the possible purpose of filamentous virus morphology and is applicable to a wide range of other biological entities, including bacteria and fungi.

Virus Detection and Identification in Minutes Using Single-Particle Imaging and Deep Learning.

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of fluorescently labeled intact particles of different viruses. Our assay achieves labeling, imaging, and virus identification in less than 5 min and does not require any lysis, purification, or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses. We were also able to differentiate closely related strains of influenza, as well as SARS-CoV-2 variants. Additional and novel pathogens can easily be incorporated into the test through software updates, offering the potential to rapidly utilize the technology in future infectious disease outbreaks or pandemics. Single-particle imaging combined with deep learning therefore offers a promising alternative to traditional viral diagnostic and genomic sequencing methods and has the potential for significant impact.

The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein.

The influenza A virus M1 mRNA is alternatively spliced to produce M2 mRNA, mRNA(3), and in some cases, M4 mRNA. Splicing of influenza mRNAs is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mRNAs encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mRNAs in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mRNAs compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mRNA.

The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes.

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.

Virus morphology: Insights from super-resolution fluorescence microscopy.

As epitomised by the COVID-19 pandemic, diseases caused by viruses are one of the greatest health and economic burdens to human society. Viruses are 'nanostructures', and their small size (typically less than 200 nm in diameter) can make it challenging to obtain images of their morphology and structure. Recent advances in fluorescence microscopy have given rise to super-resolution techniques, which have enabled the structure of viruses to be visualised directly at a resolution in the order of 20 nm. This mini-review discusses how recent state-of-the-art super-resolution imaging technologies are providing new nanoscale insights into virus structure.

NS reassortment of an H7-type highly pathogenic avian influenza virus affects its propagation by altering the regulation of viral RNA production and antiviral host response.

Highly pathogenic avian influenza viruses (HPAIV) with reassorted NS segments from H5- and H7-type avian virus strains placed in the genetic background of the A/FPV/Rostock/34 HPAIV (FPV; H7N1) were generated by reverse genetics. Virological characterizations demonstrated that the growth kinetics of the reassortant viruses differed from that of wild-type (wt) FPV and depended on whether cells were of mammalian or avian origin. Surprisingly, molecular analysis revealed that the different reassortant NS segments were not only responsible for alterations in the antiviral host response but also affected viral genome replication and transcription as well as nuclear ribonucleoprotein (RNP) export. RNP reconstitution experiments demonstrated that the effects on accumulation levels of viral RNA species were dependent on the specific NS segment as well as on the genetic background of the RNA-dependent RNA polymerase (RdRp). Beta interferon (IFN-ß) expression and the induction of apoptosis were found to be inversely correlated with the magnitude of viral growth, while the NS allele, virus subtype, and nonstructural protein NS1 expression levels showed no correlation. Thus, these results demonstrate that the origin of the NS segment can have a dramatic effect on the replication efficiency and host range of HPAIV. Overall, our data suggest that the propagation of NS reassortant influenza viruses is affected at multiple steps of the viral life cycle as a result of the different effects of the NS1 protein on multiple viral and host functions.

Functional analysis of the influenza virus H5N1 nucleoprotein tail loop reveals amino acids that are crucial for oligomerization and ribonucleoprotein activities.

Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.

Viral detection and identification in 20 min by rapid single-particle fluorescence in-situ hybridization of viral RNA.

The increasing risk from viral outbreaks such as the ongoing COVID-19 pandemic exacerbates the need for rapid, affordable and sensitive methods for virus detection, identification and quantification; however, existing methods for detecting virus particles in biological samples usually depend on multistep protocols that take considerable time to yield a result. Here, we introduce a rapid fluorescence in situ hybridization (FISH) protocol capable of detecting influenza virus, avian infectious bronchitis virus and SARS-CoV-2 specifically and quantitatively in approximately 20 min, in virus cultures, combined nasal and throat swabs with added virus and likely patient samples without previous purification. This fast and facile workflow can be adapted both as a lab technique and a future diagnostic tool in enveloped viruses with an accessible genome.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Splicing of influenza A virus NS1 mRNA is independent of the viral NS1 protein.

RNA segment 8 (NS) of influenza A virus encodes two proteins. The NS1 protein is translated from the unspliced primary mRNA transcript, whereas the second protein encoded by this segment, NS2/NEP, is translated from a spliced mRNA. Splicing of influenza NS1 mRNA is thought to be regulated so that the levels of NS2 spliced transcripts are approximately 10 % of total NS mRNA. Regulation of splicing of the NS1 mRNA has been studied at length, and a number of often-contradictory control mechanisms have been proposed. In this study, we used (32)P-labelled gene-specific primers to investigate influenza A NS1 mRNA splicing regulation. It was found that the efficiency of splicing of NS1 mRNA was maintained at similar levels in both virus infection and ribonucleoprotein-reconstitution assays, and NS2 mRNA comprised approximately 15 % of total NS mRNA in both assays. The effect of NS1 protein expression on the accumulation of viral NS2 mRNA and spliced cellular beta-globin mRNA was analysed, and it was found that NS1 protein expression reduced spliced beta-globin mRNA levels, but had no effect on the accumulation of NS2 mRNA. We conclude that the NS1 protein specifically inhibits the accumulation of cellular RNA polymerase II-driven mRNAs, but does not affect the splicing of its own viral NS1 mRNA.