Promoterless cassettes for expression of alpha virus structural proteins

1. A method of making an alphavirus replicon particle, comprising introducing one or more of an isolated RNA molecule, which RNA comprises:

a) an alphavirus 5′ replication recognition sequence, wherein at least one initiation codon has been removed from the 5′ replication recognition sequence;

b) a nucleotide sequence encoding an alphavirus structural protein; and

c) an alphavirus 3′ replication recognition sequence,

with the proviso that the RNA molecule does not contain a promoter that directs transcription of the nucleotide sequence of (b), and wherein the alphavirus 5′ and 3′ replication recognition sequences of (a) and (c) direct replication of the entire RNA molecule in the presence of alphavirus nonstructural proteins; into a cell, wherein the RNA molecule comprises: i) an alphavirus 5′ replication recognition sequence, wherein at least one initiation codon has been removed from the 5′ replication recognition sequence; ii) a nucleotide sequence encoding an alphavirus structural protein; and iii) an alphavirus 3′ replication recognition sequence, with the proviso that the RNA molecule does not contain a promoter that directs transcription of the nucleotide sequence of (ii), and wherein the alphavirus 5′ and 3′ replication recognition sequences of (i) and (iii) direct replication of the entire RNA molecule in the presence of nonstructural proteins,

whereby the combination of RNA molecules encodes all alphavirus structural proteins necessary for production of an alphavirus replicon particle, along with an alphavirus replicon RNA, under conditions whereby alphavirus replicon particles are produced.

2. The method of claim 1 , wherein two RNA molecules are introduced into the cell, wherein a first RNA molecule of the two RNA molecules encodes one or more alphavirus structural proteins and a second RNA molecule of the two RNA molecules encodes one or more alphavirus structural proteins, at least one of which is different from the alphavirus structural proteins encoded by the first RNA molecule.

3. The method of claim 1 , wherein three RNA molecules are introduced into the cell, wherein a first RNA molecule of the three RNA molecules encodes one or more alphavirus structural proteins and a second RNA molecule of the three RNA molecules encodes one or more alphavirus structural proteins, at least one of which is different from the alphavirus structural proteins encoded by the first RNA molecule and a third RNA molecule encodes one or more alphavirus structural proteins, at least one of which is different from the alphavirus structural proteins encoded by the first RNA molecule and the second RNA molecule.

4. The method of claim 1 , wherein at least one of the one or more RNA molecules is capped at the 5′ terminus.

5. A method of making an alphavirus replicon particle, comprising introducing into a cell:

a) an alphavirus replicon RNA;

b) one or more of an RNA molecule, wherein the RNA molecule comprises: i) an alphavirus 5′ replication recognition sequence, wherein at least one initiation codon has been removed from the 5′ replication recognition sequence; ii) a nucleotide sequence encoding an alphavirus structural protein; and iii) an alphavirus 3′ replication recognition sequence, with the proviso that the RNA molecule does not contain a promoter that directs transcription of the nucleotide sequence of (ii), and wherein the alphavirus 5′ and 3′ replication recognition sequences of (i) and (iii) direct replication of the entire RNA molecule in the presence of nonstructural proteins; and

c) one or more promoter-assisted alphavirus helper constructs,

whereby the combination of RNA molecules of (b) and helper constructs of (c) encodes all alphavirus structural proteins necessary for production of an alphavirus replicon particle, under conditions whereby an alphavirus replicon particle is produced.

6. The method of claim 5 , wherein at least one of the one or more RNA molecules is capped at the 5′ terminus.