Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed. comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as is compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

Abstract: A diagnostic assay for detecting a negative-strand RNA virus in a sample and a genetically engineered cell for use in the assay are disclosed. The cell expresses a heterologous DNA-dependent RNA polymerase that synthesizes a minigenome or miniantigenome of the RNA virus from a cDNA template present in the cell. The cell also expresses the nucleocapsid proteins of the negative-strand virus that are necessary for replication of the minigenome or miniantigenome. Infection of the cell by the negative-strand virus results in expression of a reporter gene product encoded by the miniantigenome.

Abstract: A recombinant cell stably transformed with a cDNA of a silent, inducible replicon encoding a recombinant protein is disclosed. Transcription of the replicon cDNA is under the control of a silent promoter inducible by a DNA virus and expression of the recombinant protein is dependent upon the presence of the DNA virus in the cell. The cell can be engineered to package the replicon upon infection by the DNA virus, leading to intercellular amplification of expression of the recombinant protein. Where the recombinant protein is a reporter gene product, the recombinant cell may be used in an assay for detecting DNA viruses. A kit for performing such an assay is also provided.

Abstract: Cell lines and methods are disclosed for detecting the presence of RNA viruses in a specimen. The cell lines are stably transformed with a DNA molecule that includes a promoter capable of being recognized by the DNA dependent RNA polymerase of the cell capable of directing the transcription of a cDNA of a structurally defective RNA virus genome operably coupled to the promoter. The cDNA contains a structural coding sequence encoding a selected reporter gene product. The RNA molecules transcribed by the DNA dependent RNA polymerase are not capable of causing the translation of the reporter gene in the cell except when an active related virus that provides the necessary trans-acting enzymes to cause the increased replication of the RNA containing the reporter gene which is then translated into the reporter gene product is provided.

Abstract: Cell lines and methods are disclosed for detecting the presence of RNA viruses in a specimen. The cell lines are stably transformed with a DNA molecule that includes a promoter capable of being recognized by the DNA dependent RNA polymerase of the cell capable of directing the transcription of a cDNA of a structurally defective RNA virus genome operably coupled to the promoter. The cDNA contains a structural coding sequence encoding a selected reporter gene product. The RNA molecules transcribed by the DNA dependent RNA polymerase are not capable of causing the translation of the reporter gene in the cell except when an active related virus that provides the necessary trans-acting enzymes to cause the increased replication of the RNA containing the reporter gene which is then translated into the reporter gene product is provided.

Abstract: Compositions and methods are disclosed for expressing heterologous coding sequences in host cells. The compositions include infectious Sindbis virus RNA molecules containing at least one heterologous coding sequence inserted within the structural region of the Sindbis virus genome. RNA molecules consisting essentially of a Sindbis virus junction region are also provided. Methods utilizing the novel compositions of the present invention to express heterologous coding sequences in transformed host cells are provided. Methods for producing infectious Sindbis virus particles containing infectious Sindbis virus RNA molecules comprising at least one heterologous coding sequence are provided.This invention was made with government support under grant numbers AI24134, AI11377 and AG05681 awarded by the National Institutes of Health. The government has certain rights in the invention.

Abstract: The present invention relates to RNA vectors comprising Sindbis virus DI RNA having inserted therein heterologous RNA. The vectors of the present invention provide for the cloning and expression of heterologous RNA in eucaryotic cells as well as the packaging of heterologous RNA into viral particles in the presence of helper virus. Transformed cells and genetically altered alphavirus particles comprising alphavirus DI RNA having inserted therein heterologous RNA are also provided.