scholarly journals Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein

2000 ◽  
Vol 74 (20) ◽  
pp. 9515-9524 ◽  
Author(s):  
Todd J. Green ◽  
Silvia Macpherson ◽  
Shihong Qiu ◽  
Jacob Lebowitz ◽  
Gail W. Wertz ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Analytical Ultracentrifugation ◽  
Structural Information ◽  
Protein Complexes ◽  
Outer Diameter ◽  
N Protein ◽  
Molar Ratios ◽  
P Protein ◽  
Vesicular Stomatitis ◽  
P Proteins

ABSTRACT To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 ± 10 Å, 78 ± 9 Å, and 83 ± 8 Å, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.

scholarly journals The Connector Domain of Vesicular Stomatitis Virus Large Protein Interacts with the Viral Phosphoprotein

2020 ◽  
Vol 94 (6) ◽  
Author(s):  
Joseph R. Gould ◽  
Shihong Qiu ◽  
Qiao Shang ◽  
Tomoaki Ogino ◽  
Peter E. Prevelige ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Structural Information ◽  
Polymerase Activity ◽  
Terminal Segment ◽  
Acceptor Site ◽  
L Protein ◽  
A Genome ◽  
Vesicular Stomatitis ◽  
Modular Layout ◽  
P Proteins

ABSTRACT Vesicular stomatitis virus (VSV) is an archetypical member of Mononegavirales, viruses with a genome of negative-sense single-stranded RNA (−ssRNA). Like other viruses of this order, VSV encodes a unique polymerase, a complex of viral L (large, the enzymatic component) protein and P (phosphoprotein, a cofactor component). The L protein has a modular layout consisting of a ring-shaped core trailed by three accessory domains and requires an N-terminal segment of P (P N-terminal disordered [PNTD]) to perform polymerase activity. To date, a binding site for P on L had not been described. In this report, we show that the connector domain of the L protein, which previously had no assigned function, binds a component of PNTD. We further show that this interaction is a positive regulator of viral RNA synthesis, and that the interfaces mediating it are conserved in other members of Mononegavirales. Finally, we show that the connector-P interaction fits well into the existing structural information of VSV L. IMPORTANCE This study represents the first functional assignment of the connector domain of a Mononegavirales L protein. Furthermore, this study localizes P polymerase cofactor activity to specific amino acids. The functional necessity of this interaction, combined with the uniqueness of L and P proteins to the order Mononegavirales, makes disruption of the P-connector site a potential target for developing antivirals against other negative-strand RNA viruses. Furthermore, the connector domain as an acceptor site for the P protein represents a new understanding of Mononegavirales L protein biology.

scholarly journals Role of Intermolecular Interactions of Vesicular Stomatitis Virus Nucleoprotein in RNA Encapsidation

2007 ◽  
Vol 82 (2) ◽  
pp. 674-682 ◽  
Author(s):  
Xin Zhang ◽  
Todd J. Green ◽  
Jun Tsao ◽  
Shihong Qiu ◽  
Ming Luo
Keyword(s):  
Crystal Structure ◽  
Intermolecular Interactions ◽  
Vesicular Stomatitis Virus ◽  
N Protein ◽  
P Protein ◽  
Intermolecular Contacts ◽  
Vesicular Stomatitis ◽  
Nucleoprotein N ◽  
Stable Ring

ABSTRACT The crystal structure of the vesicular stomatitis virus nucleoprotein (N) in complex with RNA reveals extensive and specific intermolecular interactions among the N molecules in the 10-member oligomer. What roles these interactions play in encapsidating RNA was studied by mutagenesis of the N protein. Three N mutants intended for disruption of the intermolecular interactions were designed and coexpressed with the phosphoprotein (P) in an Escherichia coli system previously described (T. J. Green et al., J. Virol. 74:9515-9524, 2000). Mutants N (Δ1-22), N (Δ347-352), and N (320-324, (Ala)5) lost RNA encapsidation and oligomerization but still bound with P. Another mutant, N (Ser290→Trp), was able to form a stable ring-like N oligomer and bind with the P protein but was no longer able to encapsidate RNA. The crystal structure of N (Ser290→Trp) at 2.8 Å resolution showed that this mutant can maintain all the same intermolecular interactions as the wild-type N except for a slight unwinding of the N-terminal lobe. These results suggest that the intermolecular contacts among the N molecules are required for encapsidation of the viral RNA.

scholarly journals Interaction of Vesicular Stomatitis Virus P and N Proteins: Identification of Two Overlapping Domains at the N Terminus of P That Are Involved in N0-P Complex Formation and Encapsidation of Viral Genome RNA

2007 ◽  
Vol 81 (24) ◽  
pp. 13478-13485 ◽  
Author(s):  
Mingzhou Chen ◽  
Tomoaki Ogino ◽  
Amiya K. Banerjee
Keyword(s):  
Complex Formation ◽  
Vesicular Stomatitis Virus ◽  
Viral Genome ◽  
Deletion Mutant ◽  
Mutant Protein ◽  
Mutant Form ◽  
N Protein ◽  
P Protein ◽  
Vesicular Stomatitis

ABSTRACT The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N0). The N0-P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N0-P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P (PΔ1-10) lacking the N-terminal 10 aa which is capable of forming the N0-P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcription-reconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PΔII+III). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N0-P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation.

scholarly journals Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

2006 ◽  
Vol 80 (13) ◽  
pp. 6368-6377 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Nayak ◽  
You Zhou ◽  
Asit K. Pattnaik
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Protein Function ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
De Novo ◽  
Virus Production ◽  
Hinge Region ◽  
Cell Periphery ◽  
P Protein ◽  
Vesicular Stomatitis

ABSTRACT The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

scholarly journals Moving the Glycoprotein Gene of Vesicular Stomatitis Virus to Promoter-Proximal Positions Accelerates and Enhances the Protective Immune Response

2000 ◽  
Vol 74 (17) ◽  
pp. 7895-7902 ◽  
Author(s):  
E. Brian Flanagan ◽  
L. Andrew Ball ◽  
Gail W. Wertz
Keyword(s):  
Immune Response ◽  
Protein Expression ◽  
Vesicular Stomatitis Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Gene Encoding ◽  
N Protein ◽  
Glycoprotein G ◽  
Vesicular Stomatitis ◽  
Negative Sense

ABSTRACT Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negative-sense RNA as its genome. The 11-kb genome encodes five genes in the order 3′-N-P-M-G-L-5′, and transcription is obligatorily sequential from the single 3′ promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3′-G-N-P-M-L-5′ (G1N2), 3′-P-M-G-N-L-5′ (G3N4), and 3′-G-P-M-N-L-5′ (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.

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