RNA virus expression and replication, methods and uses therefor

Abstract

A norovirus-permissive cell culture infected with a norovirus, and methods of culturing a norovirus, are disclosed. Norovirus-permissive cells include dendritic cell-lineage cells, and macrophage-lineage cells, such as dendritic cells, and macrophages having a deficiency in a cellular anti-viral pathway such as a STAT-1-dependent pathway, an interferon receptor-dependent pathway, or a PKR-dependent pathway. Also disclosed are methods of screening anti-viral compounds against norovirus-permissive cells infected with norovirus, and norovirus adapted to grow in fibroblasts as well as macrophages that are not deficient in a cellular anti-viral pathway. Methods of making a norovirus vaccine are also disclosed. A replicative form of norovirus as well as its use in the development of an anti-viral agent and a polypeptide expression system are also described.

1 . A method of inhibiting RNA virus replication, the method comprising contacting an RNA virus-infected cell with an inhibitor of lariat formation, wherein the RNA virus nucleic acid comprises a replicative form comprising at least one lariat comprising at least one 5′-2′ phosphodiester bond.  
     
     
         2 . The method of  claim 1 , wherein the inhibitor is a nucleobase polymer comprising a sequence selected from the group consisting of GTGAAATGA (SEQ ID NO: 10), GTGAAATGAGG (SEQ ID NO: 11), TACCGATCT (SEQ ID NO: 12), CTACCGATCTCGGG (SEQ ID NO: 13), GTGAAATGAGGTACCGAT (SEQ ID NO: 14) and a complement thereof.  
     
     
         3 . The method of  claim 1 , wherein the nucleobase polymer is a Y-shaped nucleobase polymer.  
     
     
         4 . The method of  claim 3 , wherein the nucleobase polymer comprises at least one internal L-2′-O-methyl ribopyrimidine subunit.  
     
     
         5 . The method of  claim 3 , wherein the nucleobase polymer comprises a 3′-terminal L-2′-deoxycytidine subunit.  
     
     
         6 . The method of  claim 3 , wherein the nucleobase polymer comprises an arabino-adenosine branch point.  
     
     
         7 . The method of  claim 1 , wherein the inhibitor comprises a nucleobase polymer comprising a sequence of at least about 10 contiguous nucleobases of a lariat branch point of the RNA virus nucleic acid replicative form or the complement thereof.  
     
     
         8 . The method of  claim 1 , wherein the inhibitor comprises a nucleobase polymer comprising a sequence of at least about 10 contiguous nucleobases of a sequence 5′ to a lariat branch point of the RNA virus nucleic acid replicative form or the complement thereof.  
     
     
         9 . The method of  claim 1 , wherein the inhibitor comprises a nucleobase polymer comprising a sequence of at least about 10 nucleobases of a sequence 3′ to a lariat branch point of the RNA virus nucleic acid replicative form or the complement thereof.  
     
     
         10 . The method of  claim 1 , wherein the RNA virus is a single-stranded RNA virus.  
     
     
         11 . The method of  claim 10 , wherein the RNA virus is a calicivirus.  
     
     
         12 . A method of translating a nucleic acid encoding a polypeptide, the method comprising: 
 inoculating an RNA virus-permissive cell with a viral nucleic acid which forms a lariat structure operatively linked to a sequence encoding the polypeptide; and    incubating the cell.    
     
     
         13 . The method of  claim 12 , wherein the RNA virus translation initiation sequence comprises a lariat branch point sequence.  
     
     
         14 . The method of  claim 12 , wherein the RNA virus translation initiation sequence is an RNA virus ribosome binding site.  
     
     
         15 . The method of  claim 14 , wherein the RNA virus ribosome binding site is an RNA virus internal ribosome entry site.  
     
     
         16 . The method of  claim 12 , wherein the RNA virus translation initiation sequence is a calicivirus translation initiation sequence and wherein the RNA virus-permissive cell is a calicivirus-permissive cell.  
     
     
         17 . A replicon comprising an RNA virus branch point and a sequence encoding a heterologous polypeptide.  
     
     
         18 . The replicon of  claim 17 , further comprising an RNA virus promoter.  
     
     
         19 . The replicon of  claim 18 , wherein the RNA virus branch point is a calicivirus branch point.  
     
     
         20 . The replicon of  claim 17 , wherein the RNA virus branch point comprises a sequence comprising at least 20 contiguous nucleotides having at least about 70% sequence identity with a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:161, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ED NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and portions thereof.