5′ sequence of vesicular stomatitis virus N-gene confers selective translation of mRNA

1992 ◽  
Vol 189 (3) ◽  
pp. 1585-1590 ◽  
Author(s):  
David T. Berg ◽  
Brian W. Grinnell
Keyword(s):  
Vesicular Stomatitis Virus ◽  
N Gene ◽  
Selective Translation ◽  
Vesicular Stomatitis

Frequent intragenic transcription termination within the N gene of vesicular stomatitis virus

Virology ◽  
1982 ◽  
Vol 122 (2) ◽  
pp. 239-250
Author(s):  
Balraj Singh ◽  
George B. Thornton ◽  
Jayasri Roy ◽  
Sohel Talib ◽  
Bishnu P. De ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Transcription Termination ◽  
N Gene ◽  
Vesicular Stomatitis

scholarly journals Regulation of RNA Synthesis by the Genomic Termini of Vesicular Stomatitis Virus: Identification of Distinct Sequences Essential for Transcription but Not Replication

1999 ◽  
Vol 73 (1) ◽  
pp. 297-306 ◽  
Author(s):  
Sean P. J. Whelan ◽  
Gail W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Virus ◽  
N Gene ◽  
Specific Sequence ◽  
Virus Identification ◽  
Sequence Elements ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Sequence Requirements ◽  
Transcription And Replication

ABSTRACT The RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV), a nonsegmented negative-strand RNA virus, directs two discrete RNA synthetic processes, transcription and replication. Available evidence suggests that the two short extragenic regions at the genomic termini, the 3′ leader (Le) and the complement of the 5′ trailer (TrC), contain essential signals for these processes. We examined the roles in transcription and replication of sequences in Le and TrC by monitoring the effects of alterations to the termini of subgenomic replicons, or infectious viruses, on these RNA synthetic processes. Distinct elements in Le were found to be required for transcription that were not required for replication. The promoter for mRNA transcription was shown to include specific sequence elements within Le at positions 19 to 29 and 34 to 46, a separate element at nucleotides 47 to 50, the nontranscribed leader-N gene junction. The sequence requirements for transcription within the Le region could not be supplied by sequences found at the equivalent positions in TrC. In contrast, sequences from either Le or TrC functioned well to signal replication, indicating that within the confines of the VSV termini, the sequence requirements for replication were less stringent. Deletions engineered at the termini showed that the terminal 15 nucleotides of either Le or TrC allowed a minimal level of replication. Within these confines, levels of replication were affected by both the extent of complementarity between the genomic termini and the involvement of the template in transcription. In agreement with our previous observations, increasing the extent of complementarity between the natural termini increased levels of replication, and this effect was most operative at the extreme genome ends. In addition, abolishing the use of Le as a promoter for transcription enhanced replication. These analyses (i) identified signals at the termini required for transcription and replication and (ii) showed that Le functions as a less efficient promoter for replication than TrC at least in part because of its essential role in transcription. Consequently, these observations help explain the asymmetry of VSV replication which results in the synthesis of more negative- than positive-sense replication products in infected cells.

scholarly journals A Mutant Vesicular Stomatitis Virus with Reduced Cytotoxicity and Enhanced Anterograde Trans-synaptic Efficiency

2020 ◽  
Author(s):  
Kunzhang Lin ◽  
Xin Zhong ◽  
Min Ying ◽  
Lei Li ◽  
Sijue Tao ◽  
...  
Keyword(s):  
Neural Networks ◽  
Vesicular Stomatitis Virus ◽  
Pseudorabies Virus ◽  
Brain Network ◽  
Brain Diseases ◽  
N Gene ◽  
Functional Studies ◽  
Neurotropic Viruses ◽  
Vesicular Stomatitis ◽  
And Function

Abstract Understanding the connecting structure of brain network is the basis to reveal the principle of the brain function and elucidate the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although the retrograde trans-synaptic tracing for analyzing the input neural networks with recombinant rabies and pseudorabies virus has been broadly applied in neuroscience, viral tools for analyzing the output neural networks are still lacking. The recombinant vesicular stomatitis virus (VSV) has been used for the mapping of synaptic outputs. However, several drawbacks, including high neurotoxicity and rapid lethality in experimental animals, hinder its application in long-term studies of the structure and function of neural networks. To overcome these limitations, we generated a recombinant VSV with replication-related N gene mutation, VSV-NR7A, and examined its cytotoxicity and efficiency of trans-synaptic spreading. We found that VSV-NR7A exhibits attenuated cytotoxicity, delayed but enhanced anterograde trans-synaptic tracing efficiency, compared with the wild-type VSV. Our work could provide an improved tool for structural and functional studies of neurocircuit.

scholarly journals Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice

2007 ◽  
Vol 82 (1) ◽  
pp. 207-219 ◽  
Author(s):  
David Cooper ◽  
Kevin J. Wright ◽  
Priscilla C. Calderon ◽  
Min Guo ◽  
Farooq Nasar ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immune Responses ◽  
Vesicular Stomatitis Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
N Gene ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Vesicular Stomatitis

ABSTRACT Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

scholarly journals A Mutant Vesicular Stomatitis Virus with Reduced Cytotoxicity and Enhanced Anterograde Trans-synaptic Efficiency

2020 ◽  
Author(s):  
Kunzhang Lin ◽  
Xin Zhong ◽  
Min Ying ◽  
Lei Li ◽  
Sijue Tao ◽  
...  
Keyword(s):  
Neural Networks ◽  
Vesicular Stomatitis Virus ◽  
Pseudorabies Virus ◽  
Inflammatory Responses ◽  
Time Window ◽  
Brain Diseases ◽  
N Gene ◽  
Wild Type ◽  
Animal Survival ◽  
Vesicular Stomatitis

Abstract Understanding the connecting structure of brain network is the basis to reveal the principle of the brain function and elucidate the mechanism of brain diseases. Trans-synaptic tracing with neurotropic viruses has become one of the most effective technologies to dissect the neural circuits. Although the retrograde trans-synaptic tracing for analyzing the input neural networks with recombinant rabies and pseudorabies virus has been broadly applied in neuroscience, viral tools for analyzing the output neural networks are still lacking. The recombinant vesicular stomatitis virus (VSV) has been used for the mapping of synaptic outputs. However, several drawbacks, including high neurotoxicity and rapid lethality in experimental animals, hinder its application in long-term studies of the structure and function of neural networks. To overcome these limitations, we generated a recombinant VSV with replication-related N gene mutation, VSV-NR7A, and examined its cytotoxicity and efficiency of trans-synaptic spreading. We found that by comparison with the wild-type tracer of VSV, the NR7A mutation endowed the virus lower rate of propagation and cytotoxicity in vitro, as well as significantly reduced neural inflammatory responses in vivo and much longer animal survival when it was injected into the nucleus of the mice brain. Besides, the spreading of the attenuated VSV was delayed when injected into the VTA. Importantly, with the reduced toxicity and extended animal survival, the number of brain regions that was trans-synaptically labeled by the mutant VSV was more than that of the wild-type VSV. These results indicated that the VSV-NR7A, could be a promising anterograde tracer that enables researchers to explore more downstream connections of a given brain region, and observe the anatomical structure and the function of the downstream circuits over a longer time window. Our work could provide an improved tool for structural and functional studies of neurocircuit.

scholarly journals Adding Genes to the RNA Genome of Vesicular Stomatitis Virus: Positional Effects on Stability of Expression

2002 ◽  
Vol 76 (15) ◽  
pp. 7642-7650 ◽  
Author(s):  
Gail W. Wertz ◽  
Robin Moudy ◽  
L. Andrew Ball
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Transcriptional Unit ◽  
N Gene ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Negative Strand ◽  
Transcriptional Termination ◽  
Vesicular Stomatitis

ABSTRACT Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.

scholarly journals Synergistic Attenuation of Vesicular Stomatitis Virus by Combination of Specific G Gene Truncations and N Gene Translocations

2006 ◽  
Vol 81 (4) ◽  
pp. 2056-2064 ◽  
Author(s):  
David K. Clarke ◽  
Farooq Nasar ◽  
Margaret Lee ◽  
J. Erik Johnson ◽  
Kevin Wright ◽  
...  
Keyword(s):  
Cell Culture ◽  
Vesicular Stomatitis Virus ◽  
Growth Kinetics ◽  
Lethal Dose ◽  
Reproductive Capacity ◽  
Neurological Injury ◽  
N Gene ◽  
M Gene ◽  
Vesicular Stomatitis

ABSTRACT A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their “wild-type” progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD50) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10- to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.

Sign in / Sign up

Export Citation Format

Share Document