scholarly journals The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

1999 ◽  
Vol 73 (1) ◽  
pp. 307-315 ◽  
Author(s):  
Sean P. J. Whelan ◽  
Gail W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Genomic Rna ◽  
Wild Type ◽  
Rna Sequences ◽  
Defective Interfering Particles ◽  
Cis Acting ◽  
Efficient Replication ◽  
Vesicular Stomatitis

ABSTRACT The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that acis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.

scholarly journals Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Louis-Marie Bloyet ◽  
Benjamin Morin ◽  
Vesna Brusic ◽  
Erica Gardner ◽  
Robin A. Ross ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Binding Domain ◽  
Genome Replication ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Vesicular Stomatitis ◽  
Oligomerization Domain

ABSTRACT Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD. We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

scholarly journals Overlapping Signals for Transcription and Replication at the 3′ Terminus of the Vesicular Stomatitis Virus Genome

1999 ◽  
Vol 73 (1) ◽  
pp. 444-452 ◽  
Author(s):  
Tong Li ◽  
Asit K. Pattnaik
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Virus Genome ◽  
Gene Analysis ◽  
Genomic Rna ◽  
Wild Type ◽  
Transcription Activity ◽  
Transcription Reinitiation ◽  
Vesicular Stomatitis ◽  
Negative Sense ◽  
Transcription And Replication

ABSTRACT Transcription and replication signals within the negative-sense genomic RNA of vesicular stomatitis virus (VSV) are located at the 3′ terminus. To identify these signals, we have used a transcription- and replication-competent minigenome of VSV to generate a series of deletions spanning the first 47 nucleotides at the 3′ terminus of the VSV genome corresponding to the leader gene. Analysis of these mutants for their ability to replicate showed that deletion of sequences within the first 24 nucleotides abrogated or greatly reduced the level of replication. Deletion of downstream sequences from nucleotides 25 to 47 reduced the level of replication only to 55 to 70% of that of the parental template. When transcription activity of these templates was measured, the first 24 nucleotides were also found to be required for transcription, since deletion of these sequences blocked or significantly reduced transcription. Downstream sequences from nucleotides 25 to 47 were necessary for optimal levels of transcription. Furthermore, replacement of sequences within the 25 to 47 nucleotides with random heterologous nonviral sequences generated mutant templates that replicated well (65 to 70% of the wild-type levels) but were transcribed poorly (10 to 15% of the wild-type levels). These results suggest that the minimal promoter for transcription and replication could be as small as the first 19 nucleotides and is contained within the 3′-terminal 24 nucleotides of the VSV genome. The sequences from nucleotides 25 to 47 may play a more important role in optimal transcription than in replication. Our results also show that deletion of sequences within the leader gene does not influence the site of transcription reinitiation of the downstream gene.

scholarly journals Replication and Propagation of Attenuated Vesicular Stomatitis Virus Vectors In Vivo: Vector Spread Correlates with Induction of Immune Responses and Persistence of Genomic RNA

2006 ◽  
Vol 81 (4) ◽  
pp. 2078-2082 ◽  
Author(s):  
Ian D. Simon ◽  
Jean Publicover ◽  
John K. Rose
Keyword(s):  
Immune Responses ◽  
Lymph Nodes ◽  
Vesicular Stomatitis Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Single Cycle ◽  
Pcr Assay ◽  
Vesicular Stomatitis

ABSTRACT Live-attenuated vesicular stomatitis virus (VSV) vectors expressing foreign antigens induce potent immune responses and protect against viral diseases in animal models. Highly attenuated (VSV-CT1) or single-cycle VSV (VSVΔG) vectors induce immune responses lower than those generated by attenuated wild-type VSV vectors when given intranasally. We show here that reduced spread of the more highly attenuated or single-cycle vectors to other organs, including lymph nodes, correlates with the reduction in the immune responses. A reverse transcription, real-time PCR assay for VSV genomic RNA (gRNA) sequences showed long-term persistence of gRNA from replicating vectors in lymph nodes, long after viral clearance. Such persistence may be important for induction of potent immune responses by VSV vectors.

scholarly journals Utilization of Homotypic and Heterotypic Proteins of Vesicular Stomatitis Virus by Defective Interfering Particle Genomes for RNA Replication and Virion Assembly: Implications for the Mechanism of Homologous Viral Interference

2005 ◽  
Vol 79 (15) ◽  
pp. 9588-9596 ◽  
Author(s):  
Gyoung Nyoun Kim ◽  
C. Yong Kang
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Nucleocapsid Protein ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Proteins ◽  
Genomic Rnas ◽  
Virion Assembly ◽  
Viral Interference ◽  
Vesicular Stomatitis ◽  
L Proteins

ABSTRACT Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSVInd) are capable of interfering with the replication of both homotypic VSVInd and heterotypic New Jersey serotype (VSVNJ) standard virus. In contrast, DI particles from VSVNJ do not interfere with the replication of VSVInd standard virus but do interfere with VSVNJ replication. The differences in the interfering activities of VSVInd DI particles and VSVNJ DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSVInd DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSVNJ DI particles could assemble only with homotypic VSVNJ viral proteins, although the genomic RNAs of VSVNJ DI particles could be replicated by using heterotypic VSVInd N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.

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