Filovirus vectors and noninfectious filovirus-based particles

Abstract

Cloned filovirus genomic cDNA and methods of using the cDNA are provided. Further provided are noninfectious lipid encapsulated filovirus-based particles.

1 . A method to prepare filovirus, comprising: contacting a cell with a vector comprising a promoter operably linked to a filovirus genomic cDNA or a portion thereof linked to a transcription termination sequence, a vector comprising a promoter operably linked to a DNA segment encoding a filovirus RNA transcriptase-polymerase, a vector comprising a promoter operably linked to a DNA segment encoding filovirus NP, a vector comprising a promoter operably linked to a DNA segment encoding filovirus VP30, and a vector comprising a promoter operably linked to a DNA segment encoding filovirus VP35, so as to yield infectious filovirus,
 wherein the portion of the cDNA, when transcribed, yields a RNA which is capable of being packaged into filovirus virions or which is capable of being replicated in the presence of filovirus proteins.   
     
     
         2 . The method of  claim 1  wherein the promoter in the vector comprising filovirus genomic cDNA is a RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T7 RNA polymerase promoter, or T3 RNA polymerase promoter. 
     
     
         3 . The method of  claim 1  further comprising a vector comprising a promoter operably linked to a DNA fragment of interest. 
     
     
         4 . The method of  claim 1  wherein the vector comprising the filovirus genomic cDNA further comprises a DNA fragment of interest within the genomic sequence. 
     
     
         5 . The method of  claim 1  wherein the promoter of the vector comprising the filovirus genomic cDNA is a T7 RNA polymerase promoter. 
     
     
         6 . The method of  claim 5  further comprising a vector comprising a promoter operably linked to a DNA segment encoding T7 RNA polymerase. 
     
     
         7 . The method of  claim 1  further comprising isolating the virus. 
     
     
         8 . The method of  claim 3  or  4  wherein the DNA fragment of interest encodes a detectable marker, a therapeutic protein or an immunogenic polypeptide or peptide of a pathogen or tumor antigen. 
     
     
         9 . The method of  claim 1  wherein the sequence of the genomic cDNA has one or more nucleotide deletions, insertions or substitutions relative to the sequence of a corresponding wild-type filovirus. 
     
     
         10 . A composition comprising a plurality of filovirus vectors, comprising:
 a) a vector comprising a promoter operably linked to a filovirus genomic cDNA or a portion thereof linked to a transcription termination sequence, wherein the portion of the cDNA, when transcribed, yields a RNA which is capable of being packaged into filovirus virions or which is capable of being replicated in the presence of filovirus proteins; and   b) a vector comprising a promoter operably linked to a DNA segment encoding a filovirus RNA transcriptase-polymerase, a vector comprising a promoter operably linked to a DNA segment encoding filovirus NP, a vector comprising a promoter operably linked to a DNA segment encoding filovirus VP30, and a vector comprising a promoter operably linked to a DNA segment encoding filovirus VP35.   
     
     
         11 . The composition of  claim 10  further comprising a vector comprising a promoter operably linked to a DNA fragment of interest. 
     
     
         12 . The composition of  claim 10  wherein the vector of a) further comprises a DNA fragment of interest in the same orientation as the genomic cDNA. 
     
     
         13 . The composition of  claim 11  or  12  wherein the DNA fragment of interest encodes an immunogenic polypeptide or peptide of a pathogen, a tumor antigen, or a therapeutic protein. 
     
     
         14 . The composition of  claim 10  wherein each vector of b) is on a separate plasmid. 
     
     
         15 . The composition of  claim 10  further comprising a vector comprising a promoter operably linked to a DNA segment encoding T7 RNA polymerase, wherein the promoter of the vector of a) is a T7 RNA polymerase promoter. 
     
     
         16 . The composition of  claim 10  wherein each of the vectors of b) further comprise a transcription termination sequence. 
     
     
         17 . The composition of  claim 10  wherein the cDNA is in the sense orientation. 
     
     
         18 . The composition of  claim 10  wherein the cDNA is in the antisense orientation. 
     
     
         19 . A cell contacted with the composition of  claim 10 . 
     
     
         20 . The cell of  claim 19  which is a eukaryotic cell. 
     
     
         21 . The cell of  claim 19  which is an insect, yeast, or mammalian cell. 
     
     
         22 . A vector encoding a mutant filovirus matrix protein having one or more amino acid deletions, insertions or substitutions relative to wild-type filovirus matrix protein, which mutant binds to lipid. 
     
     
         23 . The vector of  claim 22  which encodes a mutant filovirus matrix protein having residues corresponding to residues 1-276, 1-226, 50-276, 50-326 or 100-326 of the Ebola virus matrix protein. 
     
     
         24 . The vector of  claim 22  wherein the mutant filovirus matrix protein binds to the cell membrane of eukaryotic cells.