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.
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.
Background: Nucleoprotein (NP) is an essential component of the virus replication complex. Results:The crystal and quaternary structures of Lassa virus NP were determined. Conclusion: Lassa virus NP assembles into a symmetric trimer in solution.Significance: The trimeric complex may have a biological function in the virus life cycle, and its assembly could be a target for antivirals.
The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. Nand C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor ␣-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.
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.
In the present study, we have chosen a large-scale mutagenesis approach to gain insight into the structure and function of the Lassa virus RdRp domain. A total of 180 different mutants of the domain were generated by using a novel PCR-based mutagenesis technique and tested in the context of the Lassa virus replicon system. Nearly all residues, which were essential for function, clustered in the center of the three-dimensional model including the catalytic site, while residues that were less important for function mapped to the periphery of the model. The combined bioinformatics and mutagenesis data allowed deducing candidate residues for ligand interaction. Mutation of two adjacent residues in the putative palm-thumb subdomain junction, G1394 and D1395 (strain AV), led to a defect in mRNA synthesis but did not affect antigenomic RNA synthesis. In conclusion, the data provide circumstantial evidence for the existence of an RdRp domain between residues 1040 and 1540 of the Lassa virus L protein and the folding model of the domain. A functional element within the RdRp was identified, which is important for transcription but not replication of the genome.Lassa virus is a segmented negative-strand RNA virus of the family Arenaviridae. It belongs to the Old World complex of the arenaviruses, which also includes the prototype virus of the family, lymphocytic choriomeningitis virus (LCMV). Lassa virus is endemic in large areas of West Africa, where its natural reservoir host, the rodent Mastomys natalensis (23, 37), is prevalent. Transmission of the virus to humans causes Lassa fever, a hemorrhagic fever that is estimated to affect some 100,000 people annually (35). Human-to-human transmission may give rise to nosocomial epidemics with high case fatality rates (11). Currently, no vaccine exists for use in humans. Specific treatment is limited to ribavirin, a broad-spectrum nucleoside analogue, which is mainly effective during the first days of illness (34).The arenavirus genome consists of two single-stranded RNA segments, each containing two genes in opposite directions, a coding strategy called ambisense (3). The S RNA segment encodes the nucleoprotein (NP) and the glycoprotein precursor. The L RNA encodes the small matrix protein Z (41, 46) and the 200-kDa L protein (44). The minimal viral trans-acting factors required for replication and transcription of the genome are NP and L protein (19,24,27). They form, together with virus RNA, the ribonucleoprotein (RNP) complex. Although the L protein is believed to play a central role in RNA synthesis, little is known about the enzymatic function(s) of this large protein. It is most likely the source of the RNAdependent RNA polymerase (RdRp) activity associated with purified RNPs (12,15). This hypothesis is supported by sequence homology data predicting an RdRp domain between residues 1040 and 1540 of L protein (30, 49), as well as by mutational analysis of three putative catalytic residues within this domain using the LCMV replicon system (43). Structural data for the L pro...
The promoter sequences directing viral gene expression and genome replication of arenaviruses reside within the 3 and 5 termini of each RNA segment. The terminal 19 nucleotides at both ends are highly conserved among all arenavirus species and are almost completely complementary to each other. This study aimed at characterizing the Lassa virus promoter in detail. The relevance of each position in the promoter was studied by site-directed mutagenesis using the Lassa virus minireplicon system. The data indicate that the Lassa virus promoter functions as a duplex, regulates transcription and replication in a coordinated manner, and is composed of two functional elements, a sequence-specific region from residue 1 to 12 and a variable complementary region from residue 13 to 19. The first region appears to interact with the replication complex mainly via base-specific interactions, while in the second region solely base pairing between 3 and 5 promoter ends is important for promoter function.The family Arenaviridae comprises at least 23 virus species (5). Several arenaviruses, such as Lassa virus, Junin virus, Guanarito virus, Machupo virus, and lymphocytic choriomeningitis virus (LCMV), are important human pathogens. Lassa virus persists in the small rodent Mastomys natalensis, which is prevalent in sub-Saharan Africa. Transmission of the virus to humans causes Lassa fever, a life-threatening infection associated with bleeding and organ failure (13).Arenaviridae belong to the segmented negative-sense RNA viruses. The bisegmented genome consists of a small (S) and a large (L) RNA segment. Each segment contains two viral genes in opposite orientations, an arrangement called the ambisense-coding strategy (1). The S segment encodes the nucleoprotein (NP) and the glycoprotein precursor, which is posttranslationally cleaved into GP-1 and GP-2. The L segment encodes the small zinc-binding matrix protein Z and the large L protein, which contains an RNA-dependent RNA polymerase domain. NP, L protein, and viral RNA form the transcriptionally active unit, the ribonucleoprotein (RNP) complex. Both proteins are the minimal trans-acting factors required for RNA replication and transcription. Minimal cis-acting elements are the 5Ј and 3Ј noncoding regions (NCR) at the ends of the RNA segments as well as the intergenic region (13,21,23).The promoter sequences directing viral gene expression and genome replication reside within the 3Ј and 5Ј termini of each RNA segment. The terminal 19 nucleotides at both ends are highly conserved among arenaviruses and are almost completely complementary to each other. They probably hybridize, forming a panhandle structure, with the remaining part of the RNA molecule representing the circumference of the pan. This prediction is supported by electron microscopic studies (29). Recently, functional studies using the LCMV minireplicon system provided experimental evidence that the conserved termini are essential to promote replication and transcription (25). Deletion analysis showed that both 3Ј and 5Ј ...
Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1–200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure–function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.