Efficient Interaction of the Vesicular Stomatitis Virus P Protein with the L Protein or the N Protein in Cells Expressing the Recombinant Proteins

101

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.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-stranded RNA virus belonging to the rhabdovirus family. The 11,161-nucleotide genome of VSV (21) contains five genes and is encapsidated by the nucleocapsid (N) protein to form the ribonucleoprotein (RNP) complex. Associated with the RNP are the phosphoprotein (P protein) and the large polymerase subunit (L protein) (12). These three components are the transcriptionally active unit of the virus (12). The two remaining genes encode the matrix (M) protein and glycoprotein (G). The five genes are arranged in the order 3Ј N-P-M-G-L 5Ј, and the relative abundance of each individual transcript is related to its genomic position. The amounts of the individual mRNAs decrease with distance from the 3Ј end of the genome due to a single polymerase entry site at the 3Ј end of the genome (14) and localized attenuation at each gene junction (3,25,46). Thus, the N and P mRNAs are produced in the greatest abundance. The two proteins encoded by these genes form complexes in virus-infected cells (36, 39) and in vitro when expressed simultaneously (8,28,34,40). In each case, the N/P protein complexes were observed as multiple species with various molar ratios. The optimal molar ratio for efficient viral replication was found to be between 1:1 and 2:1 (N to P protein), while ratios substantially above or below this range had a negative effect on replication (28,34,36,40,47). The P protein when provided in an appropriate molar ratio to the N protein can preven...

Section: Discussionsupporting

confidence: 92%

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

Green

Macpherson²,

Qiu

et al. 2000

101

“…The fact that the main L binding domain (residues 40 to 70) would then mostly correspond to a predicted disordered region of the P protein (mapped as residues 56 to 91) (13) could be a guarantee for flexibility, and N°bound to the NH 2 end of P could serve as a chaperone for the L binding site. Finally, the L binding site in position 40 to 70/100 on P is consistent with the observation that the domain of residues 79 to 123 of the P protein of VSV is critical for binding to L (11), possibly through phosphorylation of Ser and Thr residues between residues 49 to 64 (43). Interestingly, Ser 63 to 64 of the RABV CVS strain located in a related position (Fig.…”

Section: Discussionsupporting

confidence: 85%

Peptides That Mimic the Amino-Terminal End of the Rabies Virus Phosphoprotein Have Antiviral Activity

Castel

Chtéoui

Caignard

et al. 2009

We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the aminoterminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to inhibit the transcription/replication of a rabies virus minigenome encoding luciferase in BHK-21-T7 cells; and (iii) for their capacity to inhibit rabies virus infection of BHK-21-T7 cells and of two derivatives of the neuronal SK-N-SH cell line. Peptides P60 and P57 (the first 60 and first 57 NH 2 residues of P, respectively) exhibited a rapid, strong, and long-lasting inhibitory potential on luciferase expression (>95% from 24 h to 55 h). P42 was less efficient in its inhibition level (75% for 18 to 30 h) and duration (40% after 48 h). The most promising peptides were synthesized in tandem with the Tat sequence, allowing cell penetration. Their inhibitory effects were observed on BHK-21-T7 cells infected with rabies virus and Lagos bat virus but not with vesicular stomatitis virus. In neuronal cells, a significant inhibition of both nucleocapsid inclusions and rabies virus release was observed.

“…Therefore, if N-protein conformational changes resulting from N-N interactions are required for N-P interactions, these must reside within N-terminal half of the protein. In contrast, with VSV and RV, C-terminal amino acids are required for N-P associations (30,31). Although we have not evaluated SYNV RNA binding, the P protein of VSV is required for specific binding of the N protein to viral RNA in vitro (23,24), and encapsidation of the leader RNA requires the five C-terminal residues of the N protein (6).…”

Section: Discussionmentioning

confidence: 99%

Role of the Sonchus Yellow Net Virus N Protein in Formation of Nuclear Viroplasms

Deng

Bragg²,

Ruzin³

et al. 2007

Plant rhabdoviruses are classified into the genera Cytorhabdovirus and Nucleorhabdovirus on the basis of their sites of replication, morphogenesis, and maturation (for a review, see reference 17). Sonchus yellow net nucleorhabdovirus (SYNV) replicates in the nucleus and is the most extensively characterized among the plant rhabdoviruses. SYNV encodes six genes in a negative-sense orientation; these six genes encode a nucleocapsid protein (N), a phosphoprotein (P), a putative movement protein (sc4), a matrix protein (M), a glycoprotein (G), and a large polymerase protein (L). The N, P, and L proteins are components of an infectious nucleocapsid core (15) with RNA-dependent RNA polymerase activity that can be purified from the nuclei of virus-infected cells (34,35). These core components form viroplasm-like structures within the nucleus that are thought to be the sites of viral replication (22). The N protein contains a carboxy (C)-terminal bipartite nuclear localization signal (NLS) that is required for nuclear import (7). The P protein when expressed alone localizes both inside and outside of the nucleus, but coexpression of the N and P proteins in plant and yeast (Saccharomyces cerevisiae) cells results in formation of compact subnuclear foci that are reminiscent of viroplasms (7). Sedimentation and immunological analyses have shown that in vivo associations of the N, P, and L proteins are required for RNA-dependent RNA polymerase activity (35). Yeast two-hybrid analyses and affinity chromatography experiments have also verified homologous (N-N) and heterologous (N-P) binding that is mediated by a region near the amino (N) terminus of the N protein (7). These results indicate that complex interactions of the N, P, and L proteins are required for subnuclear viroplasm formation and that the nucleocapsid cores within the viroplasms function in replication of genomic and antigenomic RNAs and in mRNA transcription (17).In the current study, we have conducted experiments to define the contributions of the N protein to the formation of viroplasms. These experiments include refined mapping to identify amino acids in an N-terminal helix-loop-helix motif of the N protein that result in subnuclear localization and N-N and N-P protein binding. We have also shown that mutations within the helix-loop-helix motif that disrupt N-N and N-P interactions interfere with the formation of subnuclear foci. The C-terminal NLS that is required for nuclear import of the N protein (7) mediates binding to importin ␣ homologues from yeast (ySrp1) and plant Arabidopsis thaliana (AtSrp1), but the P protein fails to bind to the importin ␣ homologues. These