Modified morbillivirus V proteins

A measles virus, measles virus genus technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of reduced viral transcription/replication efficiency and important function changes

Inactive Publication Date: 2007-02-07
WYETH HOLDINGS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations within any of these regions will result in changes in important functions, including decreased efficiency of viral transcription / replication

Method used

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  • Modified morbillivirus V proteins
  • Modified morbillivirus V proteins
  • Modified morbillivirus V proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1 cells and viruses

[0147] HEp2 cells were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum. Chicken embryo fibroblasts (CEF; SPAFAS, Inc) were maintained in the same medium. An attenuated strain of poxvirus expressing bacteriophage T7 RNA polymerase (MVA / T7;62) was grown in CEFs. Plaque assays were also performed on CEFs.

Embodiment 2

[0149] recombinant DNA

[0150] Measles virus N, P and L protein expression clones ( figure 1 A). Infect Vero cells with the Edmonston wild-type strain of measles virus, when about 70% or more of the cells show cytopathic effect, by - Preparation of RNA by phenol extraction (95). RT / PCR was performed with avian myovirus RT and Pwo polymerase contained in a one-tube Titan amplification kit (Roche Molecular Biology). The RT step was performed at 47°C for 30-60 minutes, followed by 30-35 rounds of PCR amplification. The amplified DNA fragment was cloned into the T7 expression plasmid ( figure 1 ;(61, 83)). Cloned DNA was checked by cycle sequencing (96) and oligonucleotide mutagenized using the Morph kit (5prime-3prime, Inc.), or corrected by subfragment replacement with newly amplified DNA fragments as previously described Nucleotide substitution errors (96).

[0151] Expression clones of the original V protein were prepared by PCR amplification from the Edmonston wil...

Embodiment 3

[0157] Transient expression experiment

[0158] As previously described (96) mainly with different amounts of viral protein expression vectors ( figure 1 B) and measles virus small replicas containing the CAT reporter gene ( figure 1 B) Transient minireplisome expression analysis was performed.

[0159] The measles virus minireplisome is a derivative of pMV107-CAT (63) containing the CAT reporter gene and the measles leader sequence (60) from the Edmonston wild-type strain of measles virus. When cells were approximately 70-90% confluent, transfect with HEp2 cells in 6-well plates. Transfection mixtures were prepared by mixing minireplisome DNA (50-200 ng pMVwt107-CAT) and expression plasmids (400 ng pMVwt-N, 300 ng pMVwt-P[C-], 100 ng pMVwt-L) in 200 microliters of serum-free OptiMEM. In this mixture according to the amount of 25-400ng, such as figure 1 Add the V protein expression plasmid as described in C. Lipofectace (12-15 microliters; Invitrogen / Life Technologies)...

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Abstract

Modified Morbilliviruses having at least one mutation in the region corresponding to amino acids 112-134 of the measles virus V protein are described wherein one or both of amino acids 113 or 114 is mutated. Such modified orbilliviruses exhibit reduced repression of gene expression. Additional mutations or deletions in other regions of the genome may be included, including in the carboxy-terminal region.

Description

field of invention [0001] The present invention relates to isolated, recombinantly produced, antisense single-stranded RNA viruses of the genus Morbillivirus having one or more mutations and / or deletions which reduce the repression normally caused by the V protein. Background of the invention [0002] Enveloped, negative-sense, single-stranded RNA viruses are uniquely composed and expressed. The genomic RNA of the negative-sense single-stranded virus serves as a template for the nucleocapsid in two ways: as a template for the synthesis of the messenger RNA (mRNA) and as a template for the synthesis of the antigenomic (+) strand. Viral replication occurs after mRNA synthesis and requires continuous synthesis of viral proteins. The newly synthesized antigenomic (+) strand serves as a template for the generation of the remaining copies of the (-) strand genomic RNA. [0003] The RNA-dependent RNA polymerase complex promotes and achieves transcription and replication by bindin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/115C12N7/00C07K14/13A61K39/165A61P31/14C12N15/09A61K39/155C07K14/12C12N7/02C12N7/04
CPCC12N7/00C12N2760/18422A61K39/165A61K39/00A61K39/155C12N2760/18461A61K2039/525A61K2039/5254C07K2319/00C07K14/005C12N2760/18434A61K39/12A61P31/14
Inventor C·L·帕克斯
Owner WYETH HOLDINGS CORP
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