Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-02-19
2001-08-07
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S030000, C435S034000, C435S325000, C435S810000, C435S975000
Reexamination Certificate
active
06270958
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention generally relates to the field of virology and, more particularly, to a method for detecting a negative-strand RNA virus in a biological specimen and genetically engineered cells for use in the method.
(2) Description of Related Art
Numerous negative-strand RNA viruses are pathogenic to humans and other animals. Examples of human diseases caused by negative-strand viruses include mumps, measles, pneumonia, bronchitis, influenza, infectious croup, rabies, ebola hemorrhagic fever, marburg hemorrhagic fever, and LaCrosse encephalitis. Thus, detection of negative-strand viruses in biological specimens is important clinically and for various research purposes.
Although there is considerable diversity in the genomic structure and biological properties of negative-strand RNA viruses, the RNA replication and transcription strategies of these viruses have common features. As with all RNA viruses, negative-strand RNA viruses express an RNA-dependent RNA polymerase and other RNA replicase and transcriptase factors necessary to transcribe their mRNA and replicate their genomes (Olivo, P. D.,
Clin. Microb. Rev.
9:321-334, 1996). In the virion of a negative-strand virus, the RNA polymerase forms a complex with the nucleocapsid protein and the genomic RNA. Once the virion enters the cell and begins to uncoat, the virion RNA polymerase transcribes subgenomic mRNAs from the genomic RNA. The RNA polymerase also synthesizes full-length positive-strand replicative-intermediate RNA (antigenome), which is used as a template for making many copies of negative-stranded genomic RNA that then are used as templates for secondary transcription of additional viral mRNAs.
The study of negative-strand viruses has been complicated by the fact that the RNA-dependent RNA polymerase uses as template only RNA associated with virus-specific nucleocapsid proteins; thus, naked genomic RNA transfected into a cell is not replicated (Bukreyev et al.,
J. Virol.
70:6634-6641, 1996; Olivo, supra). Recently, however, a number of laboratories have described advances in the genetic manipulation of various negative-strand RNA viruses. For example, infectious rabies virus, Sendai virus, vesicular stomatitis virus, measles virus, and respiratory syncytial virus (RSV) have been reportedly recovered by using T7 RNA polymerase to generate a full-length antigenomic transcript from a cDNA of the genome in the cell cytoplasm together with the viral proteins necessary for assembly of a nucleocapsid and for RNA replication and transcription (Schnell et al.,
EMBO J
13:4195-4203, 1994; Garcin et al.,
EMBO J
14:6087-6094, 1995; Lawson et al.,
Proc. Natl. Acad. Sci. USA
92:4477-4481, 1995; Radecke et al.,
EMBO J
14:5773-5784, 1995; Whelan et al.,
Proc. Natl. Acad. Sci. USA
92:8388-8392, 1995; Collins et al.,
Proc. Natl. Acad. Sci. USA
92:11563-11567, 1995). In these reports, the T7 RNA polymerase was provided to the cell cytoplasm by infection with a recombinant vaccinia virus containing the gene for T7 RNA polymerase.
Other studies investigating viral protein function in replication and transcription of negative-strand RNA viruses have employed “minigenomes”, in which some or all of the viral protein-coding sequences are replaced with a reporter gene flanked by cis-acting elements necessary for replication and transcription. See, e.g., Grosfeld et al.,
J. of Virol.
69:5677-5686, 1995; Conzelmann et al.,
J. Virol.
68:713-719, 1994; De et al.,
Virol.
196:344-348, 1993; Dimock et al.,
J. Virol.
67:27722-2778, 1993. In this approach, a cDNA is constructed in which the minigenome is operably linked to a promoter for a bacteriophage RNA polymerase, and the minigenome cDNA is transfected into cells and transcribed by the bacteriophage RNA polymerase in the presence of various viral proteins supplied by cotransfected plasmids, whose expression is also driven by the bacteriophage RNA polymerase, which is supplied by infection with a recombinant vaccinia virus. Induction of reporter gene expression indicates the transfected cell is expressing all the viral proteins needed to replicate and transcribe the minigenome.
Using this approach to study replication and transcription in RSV, the major nucleocapsid protein (N), the nucleocapsid-associated phosphoprotein (P), and the polymerase L protein were identified as sufficient to replicate the minigenome (Grosfeld et al., supra; Yu et al., J. Virol. 69:2412-2419, 1995). Coexpression of the N, P, and L proteins with a minigenome containing the cat reporter gene also resulted in synthesis of full-size and incomplete CAT mRNA species and abundant expression of CAT (Grosfeld et al., supra). In contrast, efficient synthesis of full-length mRNA was observed when cells containing the minigenome cDNA and the N, P and L expression plasmids were coinfected with the T7-expressing vaccinia virus and RSV, i.e., the minigenome RNA and the N, P and L proteins were synthesized by T7 RNA polymerase in the presence of infectious RSV (Grosfeld et al., supra). A later study demonstrated that expression of an additional RSV protein, the transcription elongation factor M2-1, was required for fully processive sequential transcription in this T7-expression plasmid complementation system (Collins et al., Proc. Natl. Acad. Sci. USA 93:81-85, 1996).
In U.S. Pat. No. 5,591,579, a genetically engineered cell line and method for detecting positive-strand RNA viruses was described. The cell line is stably transformed with a cDNA copy of a structurally defective RNA virus genome which contains (1) the cis-acting sequences of the RNA virus genome which are necessary for replication and transcription by trans-acting enzymes from the RNA virus and (2) a reporter gene. The cDNA is constitutively transcribed into a (+) strand RNA molecule from a RNA polymerase II promoter in the nucleus of the host cell, but little or no expression of the reporter gene occurs until the cell is infected by a positive-strand RNA virus that recognizes the cis-acting sequences and causes significant replication of the (+) strand RNA molecule through a (−) strand RNA intermediate.
Recently, a review article on the use of transgenic cell lines for detecting animal viruses speculated that a similar strategy could be used to make a cell line for detecting negative-strand RNA viruses (Olivo et al., supra). The article stated generally that such a cell line would constitutively express the viral nucleocapsid protein and a chimeric antigenomic RNA molecule which contains a reporter gene open reading frame (ORF) and the cis-acting sequences necessary for replication and transcription by the replicase and transcriptase of the same negative-strand RNA virus. This chimeric RNA molecule would be designed, in an unspecified manner, to preclude translation of the reporter gene in uninfected cells. Replication and transcription of the chimeric RNA molecule and synthesis of translatable reporter gene mRNA would be carried out by the replicase-transcriptase complex brought into this hypothetical cell by infection with the negative-strand RNA virus. However, this article did not teach how the nucleocapsid protein and chimeric RNA molecule would be constitutively expressed or how to design the chimeric RNA molecule to preclude translation of the reporter gene in the absence of infectious virus. It would be desirable, therefore, to provide a rapid, specific, sensitive and cost-efficient assay for the detection of infectious negative-stranded RNA viruses.
SUMMARY OF THE INVENTION
Briefly, therefore, the present invention is directed to novel compositions and methods which permit the detection of a negative-strand RNA virus in a specimen. In one embodiment, the invention provides a genetically engineered cell which expresses a reporter gene product only in the presence of infectious virions of the negative-strand RNA virus. The genetically engineered cell comprises a polynucleotide encoding a DNA-dependent RNA polymerase and a cDNA comprising a minigenome and a miniantigen
Collins Peter
Olivo Paul D.
Peeples Mark E.
Schlesinger Sondra
Howell & Haferkamp L.C.
Mosher Mary E.
Washington University
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