Modification of virus tropism and host range by viral genome shuffling

Abstract

The invention relates to a method and compositions for modifying a phenotype of a virus, such as viral tropism and host range, by iterative sequence recombination of variant viruses and selection of improved variants.

What is claimed is:  
     
         1 . A method for generating a viral polynucleotide sequence having a genotype encoding at least one modified viral phenotype, the method comprising: 
 contacting a cell or non-human animal which does not naturally support substantial replication of an predetermined virus, with at least one initial infectious virion or replicable genome of said predetermined virus under replication conditions;    recovering a plurality of replicated genome copies of said predetermined virus, either as virions or as viral genomes in polynucleotide form, wherein some or all of the replicated genome copies comprise a mutation relative to the initial infectious virion or replicable genome;    recombining a plurality of said replicated genome copies, so as to shuffle the mutations, thereby generating a collection of recombined replicated genome copies; and,    selecting or screening said collection of recombined replicated genome copies to obtain one or more replicable viral genome encoding at least one modified viral phenotype.    
     
     
         2 . The method of  claim 1 , wherein the modified viral phenotype is a host range or cell tropism phenotype.  
     
     
         3 . The method of  claim 2 , wherein the host range or cell tropism phenotype is the ability to replicate in mouse or macaque cells.  
     
     
         4 . The method of  claim 2 , wherein the host range or cell tropism phenotype is the ability to replicate in a transgenic mouse expressing a human CD4 protein or HIV co-receptor on lymphocytes.  
     
     
         5 . The method of  claim 1 , wherein the predetemined virus is selected from HIV-1, HIV-2, HCV, HBV and MLV.  
     
     
         6 . The method of  claim 5 , wherein the virus is an HIV-1 which HIV-1 is a clinical isolate which has been passaged in cell culture for less than 10 passages.  
     
     
         7 . The method of  claim 1 , wherein the modified viral phenotype is an ability to replicate in a transgenic non-human animal expressing human CD4 and human CCR5.  
     
     
         8 . The method of  claim 7 , wherein the transgenic non-human animal also expresses human CXCR4.  
     
     
         9 . The method of  claim 1 , comprising the further step of recombining a plurality of species of viral genomes of said predetermined virus, so as to shuffle the viral genome sequences, thereby generating a collection of recombined replicated genome copies, prior to contacting the viral genome with a a cell or non-human animal which does not naturally support substantial replication of said predetermined virus.  
     
     
         10 . The method of  claim 1 , which further comprises an iterative recycle of at least one interation.  
     
     
         11 . The method of  claim 9 , wherein the steps of shuffling and selection are iteratively repeated at least twice.  
     
     
         12 . A recombinant virus capable of replicating in a cell or organism which is non-permissible for replication of a wild-type virus, and made by the method of  claim 1 .  
     
     
         13 . The recombinant virus of  claim 12 , wherein the genome comprises a plurality of genome segments, wherein at least three of said genome segments are derived from nonidentical species of viral genomes.  
     
     
         14 . The recombinant virus of  claim 12 , wherein the recombinant virus has a viral genome composed of an HIV-1 genome comprising viral genome sequences from at least two clades of HIV-1.  
     
     
         15 . The recombinant virus of  claim 12 , wherein the recombinant virus is a SHIV virus.  
     
     
         16 . The recombinant virus of  claim 12 , wherein the cell or organism is a transgenic mouse cell or a transgenic mouse, said transgenic cell or transgenic mouse harboring an expressible transgene encoding human CD4.  
     
     
         17 . The recombinant virus of  claim 16 , wherein the transgenic cell or transgenic mouse further harbors an expressible transgene that encodes human CCR5.  
     
     
         18 . A selected, shuffled virus having a genotype encoding at least one modified viral phenotype.  
     
     
         19 . The selected, shuffled-virus of  claim 18 , wherein said selected shuffled virus is an HIV-1 virus or a SHIV virus and replicates in a mouse cell.  
     
     
         20 . The selected, shuffled virus of  claim 19 , wherein the mouse cell expresses human CD4 and human CCR5 encoded on a transgene or expression vector.