Structure of Rift Valley fever virus RNA-dependent RNA polymerase

2021 ◽  
Author(s):  
Xue Wang ◽  
Cuixia Hu ◽  
Wei Ye ◽  
Jia Wang ◽  
Xiaofei Dong ◽  
...  
Keyword(s):  
Protein Structure ◽  
Rna Polymerase ◽  
Drug Targets ◽  
Rift Valley Fever ◽  
Rna Synthesis ◽  
Rift Valley Fever Virus ◽  
Multiple Drug ◽  
Rna Dependent Rna Polymerase ◽  
Valley Fever ◽  
L Protein

Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus , which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus , the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5’ viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. Importance The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.

scholarly journals Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (12) ◽  
pp. 2906-2913 ◽  
Author(s):  
Benjamin Brennan ◽  
Ping Li ◽  
Richard M. Elliott
Keyword(s):  
Life Cycle ◽  
Rna Polymerase ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Rna Dependent Rna Polymerase ◽  
Valley Fever ◽  
L Protein ◽  
Infected Cells

The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family Bunyaviridae) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalyses both transcription of viral mRNAs and replication of the tripartite genome. Despite its importance, little is known about the intracellular distribution of the polymerase or its other roles during infection, primarily because of lack of specific antibodies that recognize L protein. To begin to address these questions we investigated whether the RVFV (MP12 strain) polymerase could tolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamwera virus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resulted in a polymerase that retained functionality in a minigenome assay, and we were able to rescue recombinant viruses that expressed the modified L protein by reverse genetics. The L protein could be detected in infected cells by Western blotting with anti-V5 antibodies. Examination of recombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear or cytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. The generation of RVFV expressing a tagged RdRp will allow detailed examination of the role of the viral polymerase in the virus life cycle.

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

2019 ◽  
Vol 15 (5) ◽  
pp. e1007829 ◽  
Author(s):  
Nadja Gogrefe ◽  
Sophia Reindl ◽  
Stephan Günther ◽  
Maria Rosenthal
Keyword(s):  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Binding Domain ◽  
Fever Virus ◽  
Valley Fever ◽  
L Protein

scholarly journals Modeling the Tertiary Structure of the Rift Valley Fever Virus L Protein

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1768
Author(s):  
Gideon K. Gogovi ◽  
Fahad Almsned ◽  
Nicole Bracci ◽  
Kylene Kehn-Hall ◽  
Amarda Shehu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Tertiary Structure ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Radius Of Gyration ◽  
Computational Molecular Biology ◽  
Valley Fever ◽  
L Protein

A tertiary structure governs, to a great extent, the biological activity of a protein in the living cell and is consequently a central focus of numerous studies aiming to shed light on cellular processes central to human health. Here, we aim to elucidate the structure of the Rift Valley fever virus (RVFV) L protein using a combination of in silico techniques. Due to its large size and multiple domains, elucidation of the tertiary structure of the L protein has so far challenged both dry and wet laboratories. In this work, we leverage complementary perspectives and tools from the computational-molecular-biology and bioinformatics domains for constructing, refining, and evaluating several atomistic structural models of the L protein that are physically realistic. All computed models have very flexible termini of about 200 amino acids each, and a high proportion of helical regions. Properties such as potential energy, radius of gyration, hydrodynamics radius, flexibility coefficient, and solvent-accessible surface are reported. Structural characterization of the L protein enables our laboratories to better understand viral replication and transcription via further studies of L protein-mediated protein–protein interactions. While results presented a focus on the RVFV L protein, the following workflow is a more general modeling protocol for discovering the tertiary structure of multidomain proteins consisting of thousands of amino acids.

scholarly journals Rift Valley Fever Virus L Protein Forms a Biologically Active Oligomer

2009 ◽  
Vol 83 (24) ◽  
pp. 12779-12789 ◽  
Author(s):  
Aya Zamoto-Niikura ◽  
Kaori Terasaki ◽  
Tetsuro Ikegami ◽  
C. J. Peters ◽  
Shinji Makino
Keyword(s):  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Dominant Negative ◽  
Biologically Active ◽  
Bimolecular Fluorescence Complementation ◽  
Viral Gene ◽  
Valley Fever ◽  
L Protein

ABSTRACT Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) causes mosquito-borne epidemic diseases in humans and livestock. The virus carries three RNA segments, L, M, and S, of negative or ambisense polarity. L protein, an RNA-dependent RNA polymerase, encoded in the L segment, and N protein, encoded in the S segment, exert viral RNA replication and transcription. Coexpression of N, hemagglutinin (HA)-tagged L, and viral minigenome resulted in minigenome replication and transcription, a finding that demonstrated HA-tagged L was biologically active. Likewise L tagged with green fluorescent protein (GFP) was biologically competent. Coimmunoprecipitation analysis using extracts from cells coexpressing HA-tagged L and GFP-tagged L showed the formation of an L oligomer. Bimolecular fluorescence complementation analysis and coimmunoprecipitation studies demonstrated the formation of an intermolecular L-L interaction through its N-terminal and C-terminal regions and also suggested an intramolecular association between the N-terminal and C-terminal regions of L protein. A biologically inactive L mutant, in which the conserved signature SDD motif was replaced by the amino acid residues GNN, exhibited a dominant negative phenotype when coexpressed with wild-type L in the minigenome assay system. Expression of this mutant L also inhibited viral gene expression in virus-infected cells. These data provided compelling evidence for the importance of oligomerization of RVFV L protein for its polymerase activity.

scholarly journals T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus

2008 ◽  
Vol 89 (9) ◽  
pp. 2157-2166 ◽  
Author(s):  
Matthias Habjan ◽  
Nicola Penski ◽  
Martin Spiegel ◽  
Friedemann Weber
Keyword(s):  
Rna Polymerase ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Cell Clone ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
T7 Rna Polymerase ◽  
Wild Type ◽  
Valley Fever ◽  
Pol I

Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5′ triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses.

scholarly journals Structural insights into Rift Valley fever virus replication machinery

2021 ◽  
Author(s):  
Zhongzhou Chen ◽  
Hong-Wei Wang ◽  
Xue Wang ◽  
Cuixia Hu ◽  
Jia Wang ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Protein Structures ◽  
Fever Virus ◽  
Viral Rna ◽  
Synthesis Mechanism ◽  
Valley Fever ◽  
L Protein

Abstract Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus, which accounts for over 50% of family Phenuiviridae species. RNA-dependent RNA polymerase (L protein) is responsible for facilitating the replication and transcription of the virus. We report two cryo-EM RVFV L protein structures at 3.6 Å and 3.8 Å resolution in the presence and absence of RNA, respectively. In this first L protein structure of genus Phlebovirus, viral RNA induces considerable conformational changes of the polymerase. The RVFV L protein priming loop is distinctly different from those of other L proteins and undergoes large movements related to its replication elongation role. Structural and biochemical analyses indicate that a single template can initiate RNA replication, which is notably enhanced by 5’ viral RNA. These findings advance our understanding of RNA synthesis mechanism and provide a basis for antiviral inhibitor development.

scholarly journals Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System

2005 ◽  
Vol 79 (9) ◽  
pp. 5606-5615 ◽  
Author(s):  
Tetsuro Ikegami ◽  
C. J. Peters ◽  
Shinji Makino
Keyword(s):  
Rift Valley Fever ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Rift Valley Fever Virus ◽  
Rna Replication ◽  
Valley Fever ◽  
Rna Transcripts ◽  
L Proteins ◽  
Bunyamwera Virus ◽  
Minigenome System

ABSTRACT Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-α/β) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-α/β production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

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

2019 ◽  
Author(s):  
Hanna Jérôme ◽  
Martin Rudolf ◽  
Michaela Lelke ◽  
Meike Pahlmann ◽  
Carola Busch ◽  
...  
Keyword(s):  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Regulatory Elements ◽  
Viral Transcription ◽  
Valley Fever ◽  
Replication Complex ◽  
L Protein ◽  
Minigenome System ◽  
Transcription And Replication

AbstractReplicon systems are important molecular tools for investigating the function of virus proteins and regulatory elements involved in viral RNA synthesis. We developed an ambisense minigenome system for Rift Valley fever virus (RVFV) to dissect the effects of L gene mutations on viral transcription versus replication. The S RNA segment with regulatory elements for ambisense gene expression served as backbone for the minigenome. Expression of the luciferase reporter gene allowed the overall activity of the RVFV replication complex to be assessed, while 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 active site of the cap-snatching endonuclease in the N-terminus of the L protein. Corresponding mutations led to a selective defect in the viral mRNA synthesis as described for other viruses of theBunyaviralesorder. The analysis of further L gene mutants revealed an essential and specific 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. It may be used to validate hypotheses arising from structural or biochemical investigations of the RVFV replication complex.

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