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 Immune Response in the Absence of Neurovirulence in Mice Infected with M Protein Mutant Vesicular Stomatitis Virus

2008 ◽  
Vol 82 (18) ◽  
pp. 9273-9277 ◽  
Author(s):  
Maryam Ahmed ◽  
Tracie R. Marino ◽  
Shelby Puckett ◽  
Nancy D. Kock ◽  
Douglas S. Lyles
Keyword(s):  
Central Nervous System ◽  
Immune Response ◽  
Nervous System ◽  
Vesicular Stomatitis Virus ◽  
M Protein ◽  
Wild Type ◽  
Protein Mutants ◽  
The Central Nervous System ◽  
Vesicular Stomatitis

ABSTRACT Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors.

scholarly journals Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

2002 ◽  
Vol 76 (2) ◽  
pp. 895-904 ◽  
Author(s):  
Marilyn Fernandez ◽  
Mercedes Porosnicu ◽  
Dubravka Markovic ◽  
Glen N. Barber
Keyword(s):  
Gene Therapy ◽  
Vesicular Stomatitis Virus ◽  
Mammary Adenocarcinoma ◽  
Wild Type Virus ◽  
Cytotoxic T Cell ◽  
Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Vesicular Stomatitis ◽  
Oncolytic Activity

ABSTRACT We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.

Characterization of the electrophoretic mobility mutation in the N protein of the tsD1 mutant of vesicular stomatitis virus New Jersey serotype

1982 ◽  
Vol 60 (11) ◽  
pp. 1065-1076 ◽  
Author(s):  
Earl Brown ◽  
Ludvik Prevec
Keyword(s):  
New Jersey ◽  
Electrophoretic Mobility ◽  
Vesicular Stomatitis Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Specific Chemical ◽  
N Protein ◽  
Sds Page ◽  
Vesicular Stomatitis

Some isolates of the temperature sensitive mutant tsD1 of complementation group D of vesicular stomatitis virus of New Jersey serotype have a nucleocapsid (N) protein which shows an increased electrophoretic mobility on sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS–PAGE) when compared with wild type. Utilizing techniques involving specific chemical cleavage at tryptophan or methionine residues, as well as enzymatic cleavage with carboxypeptidases A and B, we have determined that residues near the carboxyterminus are responsible for the electrophoretic difference of the mutant protein. We have further shown that there are no differences in the tryptic peptides of the mutant compared with the wild type or a non-ts revertant in this region of the protein. We have identified a tryptic peptide located outside the relevant carboxyterminal region which is distinct in mutant and revertant. We conclude that the mutation producing the aberrant electrophoretic mobility of N protein of the tsD1 mutant is a missense point mutation located at least 40 amino acid residues from the carboxyterminus and which interacts with a more proximal carboxyregion so as to influence electrophoretic mobility on SDS–PAGE.

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 Cell-Type-Specific Growth Restriction of Vesicular Stomatitis Virus polR Mutants Is Linked to Defective Viral Polymerase Function

2006 ◽  
Vol 81 (2) ◽  
pp. 492-502 ◽  
Author(s):  
Derek Ostertag ◽  
Traci M. Hoblitzell-Ostertag ◽  
Jacques Perrault
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Growth Restriction ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Protein Accumulation ◽  
Wild Type ◽  
Α Subunit ◽  
Virus Particles ◽  
Vesicular Stomatitis

ABSTRACT Vesicular stomatitis virus polR mutants synthesize defective RNA replication products in vitro and display growth restriction in some cultured cells (J. L. Chuang, R. L. Jackson, and J. Perrault, Virology 229:57-67, 1997). We show here that a recombinant virus carrying the polR N protein mutation (R179H) yielded ∼100-fold- and ∼40-fold-lower amounts of infectious virus than the wild type in mouse L-929 and rat 3Y1 cells, respectively, but only ∼3-fold less in hamster BHK cells. Virus genome accumulation was inhibited 6- to 10-fold in restricting cells, but transcription was not affected. No defect in encapsidation of replication products was detected, but virus protein accumulation was reduced two- to threefold in both restricting and nonrestricting cells. polR virus particles released from the latter were 5- to 10-fold less infectious than the wild type but showed no difference in protein composition. Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) was enhanced ∼3-fold in polR versus wild-type virus-infected L-929 cells, but neither inhibition of host gene transcription nor inhibition of double-stranded RNA (dsRNA)-activated protein kinase showed significant effects on restriction. Conditioned medium studies revealed no evidence for secretion of antiviral factors from restricting cells. We conclude that the block in polR growth is due to the combined effect of reduced genome replication and lower infectivity of released virus particles and may be due to overproduction of dsRNA. An accompanying paper (D. Ostertag, T. M. Hoblitzell-Ostertag, and J. Perrault, J. Virol. 81:503-513, 2007) provides compelling evidence for the role of dsRNA in this unique restriction phenomenon.

scholarly journals Directed attenuation to enhance vaccine immunity

2021 ◽  
Vol 17 (2) ◽  
pp. e1008602
Author(s):  
Rustom Antia ◽  
Hasan Ahmed ◽  
James J. Bull
Keyword(s):  
Immune Response ◽  
Genetic Engineering ◽  
Adaptive Immunity ◽  
Immune Evasion ◽  
Viral Infections ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Innate And Adaptive Immunity ◽  
Live Attenuated Vaccines

Many viral infections can be prevented by immunizing with live, attenuated vaccines. Early methods of attenuation were hit-and-miss, now much improved by genetic engineering. However, even current methods operate on the principle of genetic harm, reducing the virus’s ability to grow. Reduced viral growth has the undesired side-effect of reducing the host immune response below that of infection with wild-type. Might some methods of attenuation instead lead to an increased immune response? We use mathematical models of the dynamics of virus with innate and adaptive immunity to explore the tradeoff between attenuation of virus pathology and immunity. We find that modification of some virus immune-evasion pathways can indeed reduce pathology yet enhance immunity. Thus, attenuated vaccines can, in principle, be directed to be safe yet create better immunity than is elicited by the wild-type virus.

Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

1989 ◽  
Vol 92 (4) ◽  
pp. 633-642
Author(s):  
J.K. Burkhardt ◽  
Y. Argon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Intracellular Transport ◽  
Post Translational Modifications ◽  
Glycoprotein G ◽  
Golgi Compartment ◽  
Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

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