The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules.

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.

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Rift Valley Fever Virus L Protein Forms a Biologically Active Oligomer

Journal of Virology ◽

10.1128/jvi.01310-09 ◽

2009 ◽

Vol 83 (24) ◽

pp. 12779-12789 ◽

Cited By ~ 30

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.

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Structural Exploration of Rift Valley Fever Virus L Protein Domain in Implicit and Explicit Solvents by Molecular Dynamics

Advances in Computer Vision and Computational Biology - Transactions on Computational Science and Computational Intelligence ◽

10.1007/978-3-030-71051-4_59 ◽

2021 ◽

pp. 759-774

Author(s):

Gideon K. Gogovi

Keyword(s):

Molecular Dynamics ◽

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Fever Virus ◽

Protein Domain ◽

Valley Fever ◽

L Protein ◽

Implicit And Explicit

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

Journal of General Virology ◽

10.1099/vir.0.036749-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2906-2913 ◽

Cited By ~ 13

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.

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Structural insights into Rift Valley fever virus replication machinery

10.21203/rs.3.rs-398449/v1 ◽

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.

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