Recombinant alphavirus-based vectors with reduced inhibition of cellular macromolecular synthesis

A eukaryotic layered vector initiation system, comprising:

• a 5′ promoter sequence capable of initiating in vivo the 5′ synthesis of alphavirus RNA from cDNA,
• a sequence which initiates transcription of alphavirus RNA following the 5′ promoter,
• a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins,
• an alphavirus RNA polymerase recognition site, and
• a 3′ polyadenylation site,
• wherein said nucleic acid molecule encodes an altered alphaviral nonstructural protein (nsP), wherein the altered nsP is an nsP2 protein which comprises serine at the amino acid position encoded by the codon comprising nucleotide 3855 of SEQ ID NO:101.

A eukaryotic layered vector initiation system, comprising:

• a 5′ promoter sequence capable of initiating in vivo the 5′ synthesis of alphavirus RNA from cDNA,
• a sequence which initiates transcription of alphavirus RNA following the 5′ promoter,
• a nucleic acid molecule which operably encodes all four alphaviral nonstructural proteins,
• an alphavirus RNA polymerase recognition site, and
• a 3′ polyadenylation site,
• wherein said nucleic acid molecule encodes an altered alphaviral nonstructural protein (nsP), wherein the altered alphavirus nonstructural protein is an nsP4 protein which comprises histidine at the amino acid position encoded by the codon comprising nucleotide 5854 of SEQ ID NO:101.