RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment.

Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs must be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly, and budding and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA, and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z, and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for the disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment, and release in Lassa virus.

Limited high-throughput screening compatibility of the phenuivirus cap-binding domain.

Bunyaviruses constitute a large and diverse group of viruses encompassing many emerging pathogens, such as Rift Valley fever virus (family Phenuiviridae), with public and veterinary health relevance but with very limited medical countermeasures are available. For the development of antiviral strategies, the identification and validation of virus-specific targets would be of high value. The cap-snatching mechanism is an essential process in the life cycle of bunyaviruses to produce capped mRNAs, which are then recognized and translated into viral proteins by the host cell translation machinery. Cap-snatching involves cap-binding as well as endonuclease functions and both activities have been demonstrated to be druggable in related influenza viruses. Here, we explore the suitability of the phenuivirus cap-binding function as a target in medium- and high-throughput drug discovery approaches. We developed a range of in vitro assays aiming to detect the interaction between the cap-binding domain (CBD) and the analogue of its natural cap-ligand m7GTP. However, constricted by its shallow binding pocket and low affinity for m7GTP, we conclude that the CBD has limited small molecule targeting potential using classical in vitro drug discovery approaches.

Structural and functional characterization of the Sin Nombre virus L protein.

The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.

A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells.

Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.

Correction: Meier et al. Hantavirus Replication Cycle-An Updated Structural Virology Perspective. Viruses 2021, 13, 1561.

There was an error in the original publication [...].

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

The mechanism of genome replication and transcription in bunyaviruses.

Bunyaviruses are negative sense, single-strand RNA viruses that infect a wide range of vertebrate, invertebrate and plant hosts. WHO lists three bunyavirus diseases as priority diseases requiring urgent development of medical countermeasures highlighting their high epidemic potential. While the viral large (L) protein containing the RNA-dependent RNA polymerase is a key enzyme in the viral replication cycle and therefore a suitable drug target, our knowledge on the structure and activities of this multifunctional protein has, until recently, been very limited. However, in the last few years, facilitated by the technical advances in the field of cryogenic electron microscopy, many structures of bunyavirus L proteins have been solved. These structures significantly enhance our mechanistic understanding of bunyavirus genome replication and transcription processes and highlight differences and commonalities between the L proteins of different bunyavirus families. Here, we provide a review of our current understanding of genome replication and transcription in bunyaviruses with a focus on the viral L protein. Further, we compare within bunyaviruses and with the related influenza virus polymerase complex and highlight open questions.

AlphaPulldown-a python package for protein-protein interaction screens using AlphaFold-Multimer.

SUMMARY: The artificial intelligence-based structure prediction program AlphaFold-Multimer enabled structural modelling of protein complexes with unprecedented accuracy. Increasingly, AlphaFold-Multimer is also used to discover new protein-protein interactions (PPIs). Here, we present AlphaPulldown, a Python package that streamlines PPI screens and high-throughput modelling of higher-order oligomers using AlphaFold-Multimer. It provides a convenient command-line interface, a variety of confidence scores and a graphical analysis tool. AVAILABILITY AND IMPLEMENTATION: AlphaPulldown is freely available at https://www.embl-hamburg.de/AlphaPulldown. SUPPLEMENTARY INFORMATION: Supplementary note is available at Bioinformatics online.

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity.

Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.

Hantavirus Replication Cycle-An Updated Structural Virology Perspective.

Hantaviruses infect a wide range of hosts including insectivores and rodents and can also cause zoonotic infections in humans, which can lead to severe disease with possible fatal outcomes. Hantavirus outbreaks are usually linked to the population dynamics of the host animals and their habitats being in close proximity to humans, which is becoming increasingly important in a globalized world. Currently there is neither an approved vaccine nor a specific and effective antiviral treatment available for use in humans. Hantaviruses belong to the order Bunyavirales with a tri-segmented negative-sense RNA genome. They encode only five viral proteins and replicate and transcribe their genome in the cytoplasm of infected cells. However, many details of the viral amplification cycle are still unknown. In recent years, structural biology methods such as cryo-electron tomography, cryo-electron microscopy, and crystallography have contributed essentially to our understanding of virus entry by membrane fusion as well as genome encapsidation by the nucleoprotein. In this review, we provide an update on the hantavirus replication cycle with a special focus on structural virology aspects.

Reduced Nucleoprotein Availability Impairs Negative-Sense RNA Virus Replication and Promotes Host Recognition.

Negative-sense RNA viruses (NSVs) rely on prepackaged viral RNA-dependent RNA polymerases (RdRp) to replicate and transcribe their viral genomes. Their replication machinery consists of an RdRp bound to viral RNA which is wound around a nucleoprotein (NP) scaffold, forming a viral ribonucleoprotein complex. NSV NP is known to regulate transcription and replication of genomic RNA; however, its role in maintaining and protecting the viral genetic material is unknown. Here, we exploited host microRNA expression to target NP of influenza A virus and Sendai virus to ascertain how this would impact genomic levels and the host response to infection. We find that in addition to inducing a drastic decrease in genome replication, the antiviral host response in the absence of NP is dramatically enhanced. Additionally, our data show that insufficient levels of NP prevent the replication machinery of these NSVs to process full-length genomes, resulting in aberrant replication products which form pathogen-associated molecular patterns in the process. These dynamics facilitate immune recognition by cellular pattern recognition receptors leading to a strong host antiviral response. Moreover, we observe that the consequences of limiting NP levels are universal among NSVs, including Ebola virus, Lassa virus, and measles virus. Overall, these results provide new insights into viral genome replication of negative-sense RNA viruses and highlight novel avenues for developing effective antiviral strategies, adjuvants, and/or live-attenuated vaccines.IMPORTANCE Negative-sense RNA viruses comprise some of the most important known human pathogens, including influenza A virus, measles virus, and Ebola virus. These viruses possess RNA genomes that are unreadable to the host, as they require specific viral RNA-dependent RNA polymerases in conjunction with other viral proteins, such as nucleoprotein, to be replicated and transcribed. As this process generates a significant amount of pathogen-associated molecular patterns, this phylum of viruses can result in a robust induction of the intrinsic host cellular response. To circumvent these defenses, these viruses form tightly regulated ribonucleoprotein replication complexes in order to protect their genomes from detection and to prevent excessive aberrant replication. Here, we demonstrate the balance that negative-sense RNA viruses must achieve both to replicate efficiently and to avoid induction of the host defenses.

An Alcohol Dehydrogenase 3 (ADH3) from Entamoeba histolytica Is Involved in the Detoxification of Toxic Aldehydes.

Recently, a putative alcohol dehydrogenase 3, termed EhADH3B of the Entamoeba histolytica isolate HM-1:IMSS was identified, which is expressed at higher levels in non-pathogenic than in pathogenic amoebae and whose overexpression reduces the virulence of pathogenic amoebae. In an in silico analysis performed in this study, we assigned EhADH3B to a four-member ADH3 family, with ehadh3b present as a duplicate (ehadh3ba/ehadh3bb). In long-term laboratory cultures a mutation was identified at position 496 of ehadh3ba, which codes for a stop codon, which was not the case for amoebae isolated from human stool samples. When using transfectants that overexpress or silence ehadh3bb, we found no or little effect on growth, size, erythrophagocytosis, motility, hemolytic or cysteine peptidase activity. Biochemical characterization of the recombinant EhADH3Bb revealed that this protein forms a dimer containing Ni2+ or Zn2+ as a co-factor and that the enzyme converts acetaldehyde and formaldehyde in the presence of NADPH. A catalytic activity based on alcohols as substrates was not detected. Based on the results, we postulate that EhADH3Bb can reduce free acetaldehyde released by hydrolysis from bifunctional acetaldehyde/alcohol dehydrogenase-bound thiohemiacetal and that it is involved in detoxification of toxic aldehydes produced by the host or the gut microbiota.

Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein.

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

The Cap-Snatching Mechanism of Bunyaviruses.

In common with all segmented negative-sense RNA viruses, bunyavirus transcripts contain heterologous sequences at their 5' termini originating from capped host cell RNAs. These heterologous sequences are acquired by a so-called cap-snatching mechanism. Whereas for nuclear replicating influenza virus the source of capped primers as well as the cap-binding and endonuclease activities of the viral polymerase needed for cap snatching have been functionally and structurally well characterized, our knowledge on the expected counterparts of cytoplasmic replicating bunyaviruses is still limited and controversial. This review focuses on the cap-snatching mechanism of bunyaviruses in the light of recent structural and functional data.

Structure and function of the Toscana virus cap-snatching endonuclease.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

Rift Valley fever virus minigenome system for investigating the role of L protein residues in viral transcription and replication.

Replicon systems are important tools for investigating viral RNA synthesis. We have developed an ambisense minigenome system for Rift Valley fever virus (RVFV) with the aim to analyse the effects of L gene mutations on viral transcription versus replication. The overall activity of the replication complex was assessed by expression of a luciferase reporter gene. Northern blot analysis enabled differentiation between synthesis of viral mRNA and replication intermediates. The functionality of the system was demonstrated by probing residues predictably involved in the cap-snatching endonuclease active site in the L protein. Corresponding mutations led to a selective defect in the viral mRNA synthesis as described for other bunyaviruses. The analysis of further L gene mutants revealed an essential role of a C-terminal region in the RVFV L protein in viral transcription. In summary, the established minigenome system is suitable for functional testing of the relevance of residues for viral transcription and replication.

Structure of a functional cap-binding domain in Rift Valley fever virus L protein.

Rift Valley fever virus (RVFV) belongs to the family of Phenuiviridae within the order of Bunyavirales. The virus may cause fatal disease both in livestock and humans, and therefore, is of great economical and public health relevance. In analogy to the influenza virus polymerase complex, the bunyavirus L protein is assumed to bind to and cleave off cap structures of cellular mRNAs to prime viral transcription. However, even though the presence of an endonuclease in the N-terminal domain of the L protein has been demonstrated for several bunyaviruses, there is no evidence for a cap-binding site within the L protein. We solved the structure of a C-terminal 117 amino acid-long domain of the RVFV L protein by X-ray crystallography. The overall fold of the domain shows high similarity to influenza virus PB2 cap-binding domain and the putative non-functional cap-binding domain of reptarenaviruses. Upon co-crystallization with m7GTP, we detected the cap-analogue bound between two aromatic side chains as it has been described for other cap-binding proteins. We observed weak but specific interaction with m7GTP rather than GTP in vitro using isothermal titration calorimetry. The importance of m7GTP-binding residues for viral transcription was validated using a RVFV minigenome system. In summary, we provide structural and functional evidence for a cap-binding site located within the L protein of a virus from the Bunyavirales order.

Biochemical characterization of the Lassa virus L protein.

The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5'-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus.

Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses.

Viruses rely on many host cell processes, including the cellular transcription machinery. Segmented negative-strand RNA viruses (sNSV) in particular cannot synthesize the 5'-cap structure for their mRNA but cleave off cellular caps and use the resulting oligonucleotides as primers for their transcription. This cap-snatching mechanism, involving a viral cap-binding site and RNA endonuclease, is both virus-specific and essential for viral proliferation and therefore represents an attractive drug target. Here, we present biochemical and structural results on the putative cap-snatching endonuclease of Crimean-Congo hemorrhagic fever virus (CCHFV), a highly pathogenic bunyavirus belonging to the Nairoviridae family, and of two additional nairoviruses, Erve virus (EREV) and Nairobi sheep disease virus (NSDV). Our findings are presented in the context of other cap-snatching endonucleases, such as the enzymatically active endonuclease from Rift Valley fever virus (RVFV), from Arenaviridae and Bunyavirales, belonging to the His- and His+ endonucleases, respectively, according to the absence or presence of a metal ion-coordinating histidine in the active site. Mutational and metal-binding experiments revealed the presence of only acidic metal-coordinating residues in the active site of the CCHFV domain and a unique active-site conformation that was intermediate between those of His+ and His- endonucleases. On the basis of small-angle X-ray scattering (SAXS) and homology modeling results, we propose a protein topology for the CCHFV domain that, despite its larger size, has a structure overall similar to those of related endonucleases. These results suggest structural and functional conservation of the cap-snatching mechanism among sNSVs.