Methods for rescue of RNA viruses

#This invention relates to improved methods for producing nonsegmented, negative-sense, single-stranded RNA viruses of the Order designated Mononegavirales virus. Preferred embodiments relate to methods of producing such viruses as attenuated and/or infectious viruses, such as Measles virus (MV) respiratory syncytial virus (RSV) and Human parainfluenza virus (PIV). The recombinant viruses can be prepared from cDNA clones, and, accordingly, viruses having defined changes in the genome can be obtained.

#Enveloped, negative-sense, single stranded RNA viruses are uniquely organized and expressed. The genomic RNA of negative-sense, single stranded viruses serves two template functions in the context of a nucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs) and as a template for the synthesis of the antigenome (+) strand. Negative-sense, single stranded RNA viruses encode and package their own RNA-dependent RNA Polymerase. Messenger RNAs are only synthesized once the virus has entered the cytoplasm of the infected cell. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins. The newly synthesized antigenome (+) strand serves as the template for generating further copies of the (−) strand genomic RNA.

#The polymerase complex actuates and achieves transcription and replication by engaging the cis-acting signals at the 3′ end of the genome, in particular, the promoter region. Viral genes are then transcribed from the genome template unidirectionally from its 3′ to its 5′ end. There is always less mRNA made from the downstream genes (e.g., the polymerase gene (L)) relative to their upstream neighbors (i.e., the nucleoprotein gene (N)). Therefore, there is always a gradient of mRNA abundance according to the position of the genes relative to the 3′-end of the genome.

#FIG. 1 depicts a flow diagram of modified rescue procedure. This procedure includes the use of a heat shock step and transferring transfected cells to a monolayer of Vero cells.

#FIG. 2 is an autoradiogram showing the effect of heat shock on minireplicon gene expression from Example 4 through the use of CAT assays.

#FIG. 3 is an autoradiogram showing the results of CAT assays for the minireplicon RNA transfection experiments of Example 5.

#As briefly noted above, the present invention relates to a novel method of producing recombinant RNA virus. Such methods in the art are referred to as “rescue” or reverse genetics methods. Exemplary rescue methods for different nonsegmented, negative-strand viruses are disclosed in the following referenced publications: Baron and Barrett, 1997; Collins et al., 1995; Garcin et al, 1995; He et al., 1997; Hoffman and Banerjee, 1997; Lawson et al., 1995; Radecke and Billeter, 1997; Radecke et al., 1995; Schneider et al., 1997; Schnell, Mebatsion, and Conzelmann, 1994; Whelan et al., 1995. Additional publications on rescue include published International patent application WO 97/06270 for MV and other viruses of the subfamily Paramyxovirinae, and for RSV rescue, published International patent application WO 97/12032; these applications are hereby incorporated by reference.

#After transfection of a genomic cDNA plasmid, an exact copy of genome RNA is produced by the combined action of phage T7 RNA polymerase and a vector-encoded ribozyme sequence that cleaves the RNA to form the 3′ termini. This RNA is packaged and replicated by viral proteins initially supplied by co-transfected expression plasmids. In the case of the MV rescue system (Radecke et al., 1995), a stable cell line was prepared that expresses T7 RNA polymerase and the MV proteins N (nucleocapsid protein) and P (phosphoprotein). Thus, MV rescue can be achieved by co-transfecting this cell line with an MV genomic cDNA clone containing an appropriately positioned T7 polymerase promoter and an expression plasmid that contains the MV polymerase gene (L).

#One of the first few rescue methods was disclosed for Measles virus. Measles virus (MV) is a member of the Morbillivirus genus in the Paramyxoviridae family, and like all members of this family, MV is an enveloped virus that contains a nonsegmented, negative-sense RNA genome (Lamb and Kolakofsky, 1996). Molecular genetic analysis of this family of viruses has proved difficult until recently because naked genomic RNA or RNA produced intracellularly from a transfected plasmid is not infectious (Boyer and Haenni, 1994). This technical problem has been overcome through development of clever cDNA rescue technology that permits isolation of recombinant negative-strand RNA viruses (Pattnaik et al., 1992; Radecke and Billeter, 1997; Schnell, Mebatsion, and Conzelmann, 1994).