Vesicular Stomatitis Virus Nucleocapsid Protein Research Articles

Two second-site mutations compensate the engineered mutation of R7A in vesicular stomatitis virus nucleocapsid protein

The functional template for the transcription and replication of vesicular stomatitis virus (VSV) is genomic RNA encapsidated by nucleocapsid (N) protein. Previous studies showed that the amino acid R7 in the N-terminal arm of N is involved in N–N interaction in the N-RNA complex. In our study, the recombinant virus with mutation of R7A (rVSVR7A) in N was recovered, and the replication level of passage 1 (P1) of rVSVR7A was 1000 times lower than that of wild-type rVSV at 37°C. After eight passages, the replication level of P8 of rVSVR7A with two second-site mutations in the genome (T242 P in N protein and U7-U8 in G-L gene junction) was significantly higher than that of P1. Furthermore, we demonstrate that the mutation of either T242P or U7-U8 can compensate the effect caused by the mutation of R7A on the replication of rVSVR7A. Therefore, we conclude that two second-site mutations both can compensated the engineered mutation of R7A in VSV N protein.

Second-Site Mutations Selected in Transcriptional Regulatory Sequences Compensate for Engineered Mutations in the Vesicular Stomatitis Virus Nucleocapsid Protein

The active template for RNA synthesis for vesicular stomatitis virus (VSV) and other negative-strand viruses is the RNA genome in association with the nucleocapsid (N) protein. The N protein molecules sequester the genomic RNA and are linked together by a network of noncovalent interactions. We previously demonstrated that mutations predicted to weaken interactions between adjacent N protein molecules altered the levels of RNA synthesis directed from subgenomic ribonucleoprotein (RNP) templates. To determine if these mutations affect virus replication, recombinant viruses containing single-amino-acid substitutions in the N protein were recovered. Four mutations altered transcription and genome replication levels, perturbed viral protein synthesis, and inhibited virus replication. Selective pressure for improved virus replication was applied by eight sequential passages. After five passages, virus replication improved and RNA synthesis recovered concomitantly with the restoration of the protein molar ratios to near-wild-type levels. Genome sequences were compared before and after passage to determine whether compensatory mutations were selected and to potentially identify interactions between N protein molecules or between the RNP template and the viral polymerase. Improved virus replication correlated with the selection of additional mutations located in cis-acting transcriptional regulatory sequences at the gene junctions of the genome rather than in coding sequences, with one exception. The engineered N gene mutations perturbed mRNA and protein expression levels, but the selection of modified transcriptional regulatory sequences with passage facilitated the restoration of wild-type protein expression by modulating transcription levels, reflecting the adaptability and versatility of gene regulation by transcriptional control.

Plasma Membrane Microdomains Containing Vesicular Stomatitis Virus M Protein Are Separate from Microdomains Containing G Protein and Nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.

Resolution improvement of X-ray diffraction data of crystals of a vesicular stomatitis virus nucleocapsid protein oligomer complexed with RNA

Vesicular stomatitis virus (VSV) is a non-segmented negative-stranded RNA virus. The nucleocapsid (N) protein of VSV is found tightly associated with the viral genomic RNA and this complex serves as the template for transcription and replication. A method for the soluble expression of the N protein in Escherichia coli has previously been reported. An N protein-RNA oligomer was isolated from this system, the stoichiometry of which was determined to be ten molecules of the N protein bound to approximately 90 nucleotides of RNA. Here, the crystallization of this protein-nucleic acid complex is presented. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 165.65, b = 235.35, c = 75.71 A and a diffraction limit of 6 A. Self-rotation function analysis has shown the oligomer to have tenfold rotational symmetry. In a search to identify heavy-atom derivatives, uranyl acetate was discovered to stabilize the crystals, giving them an increase in diffraction limits to beyond 2.9 A. Based on the internal symmetry of the oligomer, the size of the oligomer determined previously by negative-stained electron microscopy, the space-group symmetry and packing considerations, the packing arrangement in the crystal has been determined.

RMA/S cells present endogenously synthesized cytosolic proteins to class I-restricted cytotoxic T lymphocytes.

RMA/S is a mutant cell line with decreased cell surface expression of major histocompatibility complex class I molecules that has been reported to be deficient in presenting endogenously synthesized influenza virus nucleoprotein (NP) to cytotoxic T lymphocytes (CTL). In the present study we show that RMA/S cells can present vesicular stomatitis virus nucleocapsid protein, and, under some conditions, NP, to Kb-and Db-restricted CTL, respectively. Antigen presentation results from processing of cytosolic pools of endogenously synthesized proteins, and not the binding to cell surface class I molecules of antigenic peptides present in the virus inoculum or released from infected cells. Antigen processing of RMA/S differs, however, from processing by wild-type cells in requiring greater amounts of antigen, longer times to assemble or transport class I-peptide complexes, and in being more sensitive to blocking by anti-CD8 antibody. Thus, the antigen processing deficit in RMA/S cells is of a partial rather than absolute nature.

Kinetic, quantitative, and functional analysis of multiple forms of the vesicular stomatitis virus nucleocapsid protein in infected cells.

Multiple forms of the vesicular stomatitis virus nucleocapsid protein N have been detected in infected cells. One form is complexed with the viral NS protein in a 1:1 molar ratio, and the other forms are distinguished by their more rapid sedimentation rates on glycerol gradients. I performed a series of experiments designed to analyze the relationships between these forms of the N protein. Pulse-chase experiments demonstrate that the N protein is made first as the form which binds to the NS protein, forming a 1-to-1 molar complex, and that with increasing times of chase it is either assembled into nucleocapsids or converted to the two higher sedimenting forms. Using a newly developed quantitative immunoblotting procedure, I have quantitated the three differentially sedimenting species of the N protein and have shown that at later times postinfection (6 to 7 h), the faster-sedimenting forms of the N protein account for as much as 50% of the soluble N protein in the cell. The activity of these forms has been assessed, with only the 1-to-1 molar N-NS complex demonstrating the ability to support the replication and encapsidation of viral genomic RNA. A model for the conversion of the N protein from the active N-NS complex into the other forms of the protein is presented, and the possible function of the N-protein self-complexes is discussed.

Anti-influenza virus cytotoxic T lymphocytes recognize the three viral polymerases and a nonstructural protein: responsiveness to individual viral antigens is major histocompatibility complex controlled.

It has recently been shown that antiviral major histocompatibility complex class I-restricted cytotoxic T lymphocytes can recognize proteins that serve as internal viral structural components (influenza A virus nucleoprotein, vesicular stomatitis virus nucleocapsid protein). To further examine the role of internal viral proteins in cytotoxic T-lymphocyte recognition, we constructed recombinant vaccinia viruses containing individual influenza A virus genes encoding three viral polymerases (PB1, PB2, PA) and a protein not incorporated into virions (NS1). We found that cells infected with each of these recombinant vaccinia viruses could be lysed by anti-influenza cytotoxic T lymphocytes. Cytotoxic T-lymphocyte responsiveness to the individual viral antigens varied greatly between mouse strains. By using congenic mouse strains, responsiveness to PB1 and PB2 was found to cosegregate with major histocompatibility complex haplotype. These findings provide further evidence that internal antigens play a critical role in cytotoxic T-lymphocyte recognition of virus-infected cells. Additionally, they suggest that the cytotoxic T-lymphocyte response to viral antigens may often be restricted to only a fraction of the major histocompatibility complex class I repertoire.

Vesicular stomatitis virus N and NS proteins form multiple complexes.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.

Expression of the vesicular stomatitis virus nucleocapsid protein controlled by the lambda P R and P L promoters in Escherichia coli

Expression vectors were constructed in which a cDNA specifying the vesicular stomatitis virus nucleocapsid (VSV N) protein was inserted near the translational initiation region downstream from the thermoinducible P R or P L promoter of bacteriophage λ. Expression of the VSV N-protein was determined by a radioimmunoassay with monoclonal antibody prepared against the VSV N-protein. The expression of the VSV N-protein in Escherichia coli was low with either system. However, the deletion of a part of leader sequence from the translational initiation signal to the VSV N-gene resulted in at least 30-fold increase in production of the VSV N-protein. The VSV N-protein in E. coli was also analyzed by radioimmune blot after separation of proteins by gel electrophoresis. Degraded proteins reacted with the antibody were also observed in the cell extracts.