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 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 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 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.

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 Density-Dependent Selection in Vesicular Stomatitis Virus

2004 ◽  
Vol 78 (11) ◽  
pp. 5799-5804 ◽  
Author(s):  
Isabel S. Novella ◽  
Daniel D. Reissig ◽  
Claus O. Wilke
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Resistant Mutant ◽  
Relative Fitness ◽  
Wild Type ◽  
Common Pool ◽  
Model Predictions ◽  
A Cell ◽  
Vesicular Stomatitis ◽  
Average Fitness ◽  
Good Agreement

ABSTRACT We used vesicular stomatitis virus to test the effect of complementation on the relative fitness of a deleterious mutant, monoclonal antibody-resistant mutant (MARM) N, in competition with its wild-type ancestor. We carried out competitions of MARM N and wild-type populations at different multiplicities of infection (MOIs) and initial ratios of the wild type to the mutant and found that the fitness of MARM N relative to that of the wild type is very sensitive to changes in the MOI (i.e., the degree of complementation) but depends little, if at all, on the initial frequencies of MARM N and the wild type. Further, we developed a mathematical model under the assumption that during coinfection both viruses contribute to a common pool of protein products in the infected cell and that they both exploit this common pool equally. Under such conditions, the fitness of all virions that coinfect a cell is the average fitness in the absence of coinfection of that group of virions. In the absence of coinfection, complementation cannot take place and the relative fitness of each competitor is only determined by the selective value of its own products. We found good agreement between our experimental results and the model predictions, which suggests that the wild type and MARM N freely share all of their gene products under coinfection.

scholarly journals Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

1996 ◽  
Vol 135 (1) ◽  
pp. 153-167 ◽  
Author(s):  
J Peränen ◽  
P Auvinen ◽  
H Virta ◽  
R Wepf ◽  
K Simons
Keyword(s):  
Epithelial Cells ◽  
Membrane Transport ◽  
Vesicular Stomatitis Virus ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Wild Type ◽  
Expression Systems ◽  
Vesicular Stomatitis

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

scholarly journals B7 Costimulation Is Critical for Antibody Class Switching and CD8+ Cytotoxic T-Lymphocyte Generation in the Host Response to Vesicular Stomatitis Virus

2000 ◽  
Vol 74 (1) ◽  
pp. 203-208 ◽  
Author(s):  
Alexander J. McAdam ◽  
Evan A. Farkash ◽  
Benjamin E. Gewurz ◽  
Arlene H. Sharpe
Keyword(s):  
Viral Infection ◽  
Vesicular Stomatitis Virus ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Class I ◽  
Specific Class ◽  
Wild Type ◽  
B7 Molecules ◽  
Vesicular Stomatitis ◽  
Ctl Responses

ABSTRACT Antibody and cytotoxic T-lymphocyte (CTL) responses have critical roles in eliminating many viral infections. In addition to stimulation of the T-cell receptor, T cells require costimulatory signals to respond optimally. We evaluated the role of B7 costimulatory molecules (B7-1 and B7-2) in the immune response to viral infection using vesicular stomatitis virus (VSV) and mice lacking either B7-1 or B7-2 or both molecules. Mice lacking both B7-1 and B7-2 had essentially no anti-VSV immunoglobulin G1 (IgG1) response, decreased IgG2a responses, and normal IgM responses, while mice lacking either B7-1 or B7-2 had unaltered anti-VSV antibody responses compared to wild-type mice. Depletion of CD4+ cells further reduced the IgG2a response in mice lacking both B7 molecules, suggesting that CD4−cells may supply help for IgG2a in the absence of B7 costimulation. The absence of both B7 molecules profoundly reduced generation of both primary and secondary VSV-specific class I major histocompatibility complex (MHC)-restricted CTL, whereas VSV-specific CTL responses in mice lacking either B7-1 or B7-2 were similar to those of wild-type animals. Class I MHC-restricted CTL in wild-type mice were not dependent on CD4+ cells, suggesting that the failure of CTL in the absence of B7s is due to a lack of B7 costimulation directly to the CD8+ CTL. These data demonstrate that B7-1 and B7-2 have critical, overlapping functions in the antibody and CTL responses to this viral infection.

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