Stabilization of Vesicular Stomatitis Virus L Polymerase Protein by P Protein Binding: A Small Deletion in the C-Terminal Domain of L Abrogates Binding

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.

Section: Discussionmentioning

confidence: 99%

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

Morin

Liang

Gardner

et al. 2017

“…P is a multifunctional protein: it associates with newly synthesized N during infection to prevent N from binding to cellular RNAs and maintains the replication-competent form of N by specifically encapsidating the nascent viral genomic/antigenomic RNA (4)(5)(6). On the other hand, it may also help to protect the L protein from proteolytic degradation (7). In addition, P also forms a tripartite replicase complex with L and N (L-N-P) to convert the N-RNA template to positive-sense genomic RNA in vivo.…”

mentioning

confidence: 99%

N-Terminal Phosphorylation of Phosphoprotein of Vesicular Stomatitis Virus Is Required for Preventing Nucleoprotein from Binding to Cellular RNAs and for Functional Template Formation

Chen

Zhang

Banerjee

et al. 2013

bThe phosphoprotein (P) of vesicular stomatitis virus (VSV) plays essential roles in viral RNA synthesis. It associates with nascent nucleoprotein (N) to form N 0 -P (free of RNAs), thereby preventing the N from binding to cellular RNAs and maintaining the N in a viral genomic RNA encapsidation-competent form for transcription and replication. The contributions of phosphorylation of P to transcription and replication have been studied intensively, but a concrete mechanism of action still remains unclear. In this study, using a VSV minigenome system, we demonstrated that a mutant of P lacking N-terminal phosphorylation (P3A), in which the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), does not support transcription and replication. However, results from protein interaction assays showed that P3A self-associates and interacts with N and the large protein (L) as efficiently as P does. Furthermore, purified recombinant P3A from Sf21 cells supported transcription in an in vitro transcription reconstitution assay. We also proved that P3A is not distributed intranuclearly in vivo. CsCl gradient centrifugation showed that P3A is incapable of preventing N from binding to cellular RNAs and therefore prevents functional template formation. Taken together, our results demonstrate that N-terminal phosphorylation is indispensable for P to prevent N from binding to nonviral RNAs and to maintain the N-specific encapsidation of viral genomic RNA for functional template formation. V esicular stomatitis virus (VSV) is a nonsegmented negativestrand RNA virus that serves as the prototype of the rhabdovirus group. The active RNA polymerase complex of VSV is composed of the large protein (L) and its cofactor, the phosphoprotein (P) (1), and both are required for the transcription and replication of the viral negative-sense single-stranded RNA genome, which is tightly encapsidated by nucleoprotein (N) (2, 3). P is a multifunctional protein: it associates with newly synthesized N during infection to prevent N from binding to cellular RNAs and maintains the replication-competent form of N by specifically encapsidating the nascent viral genomic/antigenomic RNA (4-6). On the other hand, it may also help to protect the L protein from proteolytic degradation (7). In addition, P also forms a tripartite replicase complex with L and N (L-N-P) to convert the N-RNA template to positive-sense genomic RNA in vivo. Via mutational and biochemical studies, P of the VSV Indiana serotype has been divided arbitrarily into three domains, i.e., N-terminal domain I (residues 1 to 137), domain II (residues 211 to 244), and domain III (residues 245 to 265), and a hypervariable hinge region (residues 138 to 210).Like the P's of many other negative-strand RNA viruses, the P of VSV is phosphorylated in infected cells and virions (8-10). The P of the VSV Indiana serotype has been shown to be phosphorylated in two different domains: N-terminal domain I, in which phosphorylation sites were mapped to S60, T62, and S64, and ...

“…P protein, on the other hand, acts as an accessory subunit of the viral polymerase (1,45). In addition to having a role in polymerase functions, it binds to the L protein and stabilizes it from proteolytic degradation (7,17), it complexes with the newly synthesized N protein for the efficient encapsidation of nascent RNA (16,36,42), and it interacts with terminal sequences of viral genome for viral RNA synthesis (29,30).…”

mentioning

confidence: 99%

Role of the Hypervariable Hinge Region of Phosphoprotein P of Vesicular Stomatitis Virus in Viral RNA Synthesis and Assembly of Infectious Virus Particles

Das

Pattnaik

2005

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNAdependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using a series of deletion mutants spanning the hinge region of the protein, we observed that amino acid residues 201 through 220 are required for the activity of P protein in both replication and transcription. Neither insertion nor deletion had any effect on the interaction of P protein with N or L proteins. Infectious VSVs with a deletion in the hinge region possessed retarded growth characteristics and exhibited small-plaque morphology. Interestingly, VSV containing one P protein deletion mutant (P⌬7, with amino acids 141 through 200 deleted), which possessed significant levels of replication and transcription activity, could be amplified only by passage in cells expressing the wild-type P protein. We conclude that the hypervariable hinge region of the P protein plays an important role in viral RNA synthesis. Furthermore, our results provide a previously unidentified function for the P protein: it plays a critical role in the assembly of infectious VSV.Vesicular stomatitis virus (VSV) is the prototypic member of the family Rhabdoviridae in the order Mononegavirales. VSV is an enveloped virus with a negative-stranded RNA genome of 11,161 nucleotides. The viral envelope consists of a lipid bilayer with the spike glycoprotein protruding out and the matrix (M) protein lying underneath the lipid bilayer. The viral genome which is present in the virion core is tightly encapsidated by the nucleocapsid protein (N protein) to form the ribonucleoprotein complex (6). The viral ribonucleoprotein serves as the template for transcription and replication by the viral RNA-dependent RNA polymerase, which is a complex of the large protein (L protein) and phosphoprotein (P protein). The L protein is the catalytic subunit of the polymerase complex responsible for polymerization of nucleotides, capping, methylation of capped mRNAs, and polyadenylation of mRNAs (reviewed in references 1 and 45). P protein, on the other hand, acts as an accessory subunit of the viral polymerase (1, 45). In addition to having a role in polymerase functions, it binds to the L protein and stabilizes it from proteolytic degradation (7, 17), it complexes with the newly synthesized N protein for the efficient encapsidation of nascent RNA (16,36,42), and it interacts with terminal sequences of viral genome for viral RNA synthesis (29,30).The P proteins of the two s...