Interactions amongst rabies virus nucleoprotein, phosphoprotein and genomic RNA in virus-infected and transfected cells

Liu, Pinghua; Yang, Jun; Wu, Xianfu; Fu, Zhen F.

doi:10.1099/vir.0.80325-0

Cited by 28 publications

(21 citation statements)

References 24 publications

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“…Disrupting phosphoprotein-nucleoprotein interactions leads to defects in viral-RNA replication and/or transcription, presumably through the loss of proper nucleocapsid formation (5). However, the current hypothesis does not explain why nonspecific RNAs are not packaged into virions, as NP associates with nonviral RNA (18,32). Our data suggest that VP35 specifically interacts with RNA.…”

Section: Discussionmentioning

confidence: 40%

“…However, little is known about the role of VP35 in the assembly of nucleocapsids or the specific encapsulation of viral RNA by the nucleocapsid and the nucleocapsid's subsequent assembly into a mature virion. The current hypothesis is that phosphoprotein and nucleoprotein interactions link nucleoprotein-RNA complexes with phosphoprotein-polymerase complexes to form viral nucleocapsids (18,27,32). The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging into virions.…”

Section: Discussionmentioning

confidence: 99%

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Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

Johnson

McCarthy

Godlewski

et al. 2006

J Virol

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The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.

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Section: Discussionmentioning

confidence: 40%

Section: Discussionmentioning

confidence: 99%

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

Johnson

McCarthy

Godlewski

et al. 2006

J Virol

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“…N encapsidates the RNA genome in a tight helical RNase-resistant ribonucleoprotein (RNP) capsid (29). P has been shown to associate with N (30, 31) before RNP capsid assembly at a P:N ratio of 2:1, in contrast to the 1:2 ratio found in the completed capsid (32)(33)(34). Formation of the RNP capsid is followed by virion assembly and budding, models for which can be found in the literature (29).…”

mentioning

confidence: 97%

Host–rabies virus protein–protein interactions as druggable antiviral targets

Lingappa¹,

Macieik³

et al. 2013

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

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We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a hostcatalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.assembly intermediate | viral-host interaction | whole pathway screen | drug discovery paradigm | protein heterogeneity

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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).…”

mentioning

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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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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Interactions amongst rabies virus nucleoprotein, phosphoprotein and genomic RNA in virus-infected and transfected cells

Cited by 28 publications

References 24 publications

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

Host–rabies virus protein–protein interactions as druggable antiviral targets

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

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