Rabies virus chaperone: Identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA

Mavrakis, Manos; Méhouas, Stéphanie; Réal, Éléonore; Iseni, Frédéric; Blondel, Danielle; Tordo, Noël; Ruigrok, Rob W.H.

doi:10.1016/j.virol.2006.01.030

Cited by 93 publications

(94 citation statements)

References 22 publications

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“…Like VSV P, RABV P comprises three domains: a C-terminal domain (P CTD ) that binds the genomic N-RNA, a central oligomerization domain (P OD ), and an N-terminal domain (P NTD ) that binds L (5,(17)(18)(19). During replication of the template, the P NTD also engages N to keep it soluble (N 0 ) for loading onto the nascent RNA chain concomitant with its synthesis by L (18,(20)(21)(22)(23)(24)(25)(26)(27).…”

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An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity

Morin

Liang

Gardner

et al. 2017

J Virol

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We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. IMPORTANCEThe current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the fulllength L protein of RABV and the characterization of its RdRP activity in vitro. The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.

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confidence: 99%

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity

Morin

Liang

Gardner

et al. 2017

J Virol

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“…It is thought that NPs must also exist in an auto-inhibited form to avoid RNA binding or oligomerization in the wrong biological context. This can occur by intra-, rather than intermolecular, binding of the flexible arms [e.g., RVFV (16), influenza (17)], rotation of an entire subdomain [e.g., CCHFV stalk domain (13,14)], or chaperoning by another protein [e.g., the phosphoprotein in nsNSVs (18)]. …”

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Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein

Reguera

Malet

Weber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged N-and C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.protein-RNA interactions | structural biology T he largest family of segmented negative-strand RNA viruses (sNSVs), Bunyaviridae, comprises more than 350 species belonging to five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus. Bunyaviruses generally infect mammals, except Tospoviruses that infect plants, and use arthropods as vectors for their spread, although Hantaviruses are rodent borne (1, 2). Several species can cause severe zoonotic diseases in humans such as La Crosse Orthobunyavirus (LACV, childhood encephalitis), Rift Valley Fever Phlebovirus (RVFV), and Crimean Congo Haemorrhagic Fever Nairovirus (CCHFV). As vector-borne diseases, their geographical occurrence is closely linked to the environment, and climate change or other ecological factors may lead to altered distribution or emergence of new viruses (3-5).Bunyaviruses have a trisegmented negative-strand RNA genome. The L segment encodes for the RNA-dependent RNA polymerase or L protein, the M segment for glycoproteins and a nonstructural protein, and the S segment encodes the nucleoprotein (NP) and another nonstructural protein. The genome is always coated by multiple copies of NP forming ribonucleoprotein particles (RNPs), which also contain the viral polymerase. RNPs are the only form under which the genome is efficiently replicated and transcribed, and NP and L are necessary and sufficient to perform these functions. Like the polymerase, which catalyses RNA replication and transcription by cap-snatching (6), ...

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“…During replication of many negative-strand RNA viruses, the nascent nucleoprotein (usually termed N) is bound by a polymerase cofactor (often a phosphoprotein, termed P), which prevents polymerization of N and nonspecific encapsidation of host cell RNAs (12)(13)(14)(15). The resulting complex is termed N 0 -P, in which N 0 denotes RNA-free N. The arenavirus, orthomyxovirus (flu), and bunyavirus (Hanta, Rift Valley Fever) families (i.e., segmented NSV) do not encode an analogous P protein, and the mechanism by which the nucleoprotein controls RNA binding during virus infection is not yet understood.…”

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Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

Hastie¹,

Liu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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165

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Arenaviruses cause disease in industrialized and developing nations alike. Among them, the hemorrhagic fever virus Lassa is responsible for ∼300,000-500,000 infections/y in Western Africa. The arenavirus nucleoprotein (NP) forms the protein scaffold of the genomic ribonucleoprotein complexes and is critical for transcription and replication of the viral genome. Here, we present crystal structures of the RNA-binding domain of Lassa virus NP in complex with ssRNA. This structure shows, in contrast to the predicted model, that RNA binds in a deep, basic crevice located entirely within the N-terminal domain. Furthermore, the NP-ssRNA structures presented here, combined with hydrogen-deuterium exchange/MS and functional studies, suggest a gating mechanism by which NP opens to accept RNA. Directed mutagenesis and functional studies provide a unique look into how the arenavirus NPs bind to and protect the viral genome and also suggest the likely assembly by which viral ribonucleoprotein complexes are organized.structural biology | virology T he arenavirus family has a worldwide distribution and contains significant human pathogens such as Lassa (LASV), Machupo, Junin, Lujo (1, 2), and lymphyocytic choriomeningitis virus. Of these arenaviruses, LASV carries the largest disease burden, causing 300,000 to 500,000 infections per year in Western Africa. It is also the hemorrhagic fever most frequently transported out of Africa to the United States and Europe (2-4).Arenaviruses have a bisegmented, negative-sense, singlestranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) (2). Of these proteins, NP is the most abundant in an infected cell. NP associates with L to form the ribonucleoprotein (RNP) core for RNA replication and transcription (5) and the matrix protein Z for viral assembly (6-8). The arenavirus NP also plays an important role in the suppression of the innate immune system (9-11).Genome and antigenome RNAs of negative-strand RNA viruses (NSV) do not exist as naked RNA, but rather as a RNP complex in which the RNA is encapsidated by the viral nucleoprotein. During replication of many negative-strand RNA viruses, the nascent nucleoprotein (usually termed N) is bound by a polymerase cofactor (often a phosphoprotein, termed P), which prevents polymerization of N and nonspecific encapsidation of host cell RNAs (12-15). The resulting complex is termed N 0 -P, in which N 0 denotes RNA-free N. The arenavirus, orthomyxovirus (flu), and bunyavirus (Hanta, Rift Valley Fever) families (i.e., segmented NSV) do not encode an analogous P protein, and the mechanism by which the nucleoprotein controls RNA binding during virus infection is not yet understood.The arenavirus nucleoprotein (termed NP instead of N) has distinct N-and C-terminal domains connected by a flexible linker (16)(17)(18)(19). The C-terminal domain functions as an exonuclease (16, 17) specific for dsRNA (17) and linked to antagonism of t...

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Rabies virus chaperone: Identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA

Cited by 93 publications

References 22 publications

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity

Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein

Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

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