Nodavirus-like DNA expression vector and uses thereof

Details Nodavirus-like DNA expression vector and uses thereof Nodavirus-like DNA expression vector and uses thereof

2000-06-15

2003-02-04

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C536S023100, C536S023720

Reexamination Certificate

active

06514757

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the molecular biology of nodaviruses. More specifically, the present invention relates to a nodavirus-based DNA expression vector, in which nodaviral RNA replicases and nodaviral cis-elements amplify a transcript containing a heterologous gene by RNA replication.
2. Description of the Related Art
The nodaviruses are a family of small icosahedral viruses with bipartite, single-stranded, positive-sense RNA genomes. Several different members of the virus family have been isolated from insects and fish larvae, but none from higher animals. However, despite the limited natural host ranges of the viruses, many nodavirus RNA replicase enzymes retain full activity in insect, mammalian, plant, and even yeast cells, and they are among the simplest and most powerful eukaryotic RNA replication enzymes known. Replication by nodavirus RNA replicase enzymes produces large amounts of capped mRNAs in the cytoplasm; for example, after amplification of nodamura virus (NoV) RNAs in baby hamster kidney (BHK21) cells for 24 hours, positive-sense RNA replication products approximate the abundance of ribosomes (about 10
7
molecules per cell) and dominate the cell's capacity for protein synthesis.
The molecular biology of the Nodaviridae is summarized schematically in
FIG. 1
(1,2). Virus particles contain one molecule each of two single-stranded genome segments, RNA1 and RNA2, both of which are required for viral infectivity. These RNAs have 5′ caps but no 3′ polyadenylate tails. Instead, their 3′ ends are blocked by either a covalent modification or an unusual secondary structure. RNA1, which in the well-characterized flock house virus (FHV), contains 3107 nucleotides (nt), encodes the entire viral contribution to the RNA-dependent RNA polymerase (RNA replicase) which replicates both RNA1 and RNA2 genome segments. RNA1 can therefore replicate independently of RNA2 and constitutes an autonomous RNA replicon (3). Its nucleotide sequence shows an open reading frame (ORF) which predicts a 112 kilodalton (kDa) polypeptide (protein A), corresponding to the catalytic subunit of the RNA replicase. During replication of RNA1, a sub-genomic RNA (RNA3), which represents the 3′ 387 nt of RNA1, is produced by partial transcription (4). RNA3 encodes, in overlapping reading frames, two small, non-structural proteins of about 11 kDa each (B1 and B2). The functions of these proteins are unknown, and neither appears to be essential for RNA replication.
Flock house virus (FHV) RNA2 contains 1400 nucleotides and encodes protein &agr;, a 45 kDa precursor of the two viral capsid proteins &bgr; (40 kDa) and &ggr; (5 kDa) (5). 180 copies of protein a assemble with T=3 icosahedral symmetry around RNA1 and RNA2, forming provirions that co-sediment with mature virus particles but lack infectivity (6). Particle assembly triggers the self-cleavage of &agr;, which yields the mature capsid proteins &bgr; and &ggr;. This cleavage stabilizes the particles and renders them infectious (7,8).
It is clear that the nodaviruses, with genomes of only 4.5 kb, are among the simplest of all animal viruses. Nevertheless, their RNAs can replicate abundantly in a wide variety of intracellular environments from yeast to mammalian cells, implying that the necessary host cell factors are conserved. The fact that some nodaviruses can readily establish persistent infections of cells in culture (1,2) indicates that long-term replication of viral RNA is not necessarily cytotoxic. Furthermore, the segmented structure of the nodaviral genome simplifies experimental manipulation of the replicase gene and shows that the RNA replicase functions naturally in trans, a convenient property for its use in an expression vector.
Most expression vectors use DNA-templated transcription to achieve the intracellular accumulation of RNAs, a process that is linearly dependent upon the copy number of the DNA template. The properties of nodaviruses and their RNA replicases create a n attractive opportunity for the development of a new type of expression vector. It is the goal of the present invention to provide a DNA plasmids which are initially transcribed by a host-cell RNA polymerase to produce primary transcripts from which the RNA replicase is translated. These primary transcripts are then amplified by autonomous, cytoplasmic, RNA replication. The performance of these vectors depends less on the efficiency of primary transcription than on the exponential amplification of the primary transcripts by nodaviral RNA replication. As described above, this process occurs abundantly in a very broad range of host cells and is largely independent of the level of cellular metabolic activity. These plasmid vectors should therefore be suitable for use in insect, mammal, plant and yeast cells, and they may also offer advantages in quiescent cells in which conventional expression vectors oftentimes fail.
The prior art is deficient in the availability of nodavirus-based expression vectors that are amplified autonomously in a cell by RNA replication. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
It is a goal of the present invention to provide a nodavirus-based DNA expression vector that is capable of yielding high levels of recombinant transcripts and/or proteins in a wide spectrum of cell types. The vector contains a promoter site for aDNA-dependent RNA polymerase to drive the synthesis of a primary transcript. The primary transcript minimally contains two sequence elements. At the 5′ end, the primary RNA transcript contains an open reading frame for a catalytic subunit of an RNA-dependent RNA polymerase (RNA replicase) from a nodavirus, preferably flock house virus (FHV), Nodamura virus (NoV) or Pariacoto virus (PaV). Translation of the 5′ open reading frame of the chimeric primary transcript in cells transfected with the nodavirus-based DNA expression vector using host-cell machinery produces an active nodavirus RNA-dependent RNA polymerase, which subsequently recognizes and replicates the primary transcript. Additionally included in the primary RNA transcript are the nodaviral cis-acting signals required for recognition by the RNA replicase. The cis-acting signals flank, at least, the heterologous gene that encodes a desirable transcript and/or protein (e.g., a ribozyme, an antisense RNA, or a protein with therapeutic potential) such that the transcript encoded by the heterologous gene is amplified by RNA replication. Preferably, cis-elements are positioned such that the transcript encoding the RNA replicase is also replicated and subsequently amplified. This strategy produces abundant levels of capped, functional mRNAs in the cytoplasm which are readily expressed by the host cell. Attractive features of this novel type of expression vector are its high expression levels and broad host range.
One object of the present invention is to provide a nodavirus-based DNA expression vector.
In an embodiment of the present invention, there is provided an isolated cDNA encoding a Nodamura virus (NoV) RNA1 genome segment having the sequence shown in SEQ D No. 1; an isolated cDNA encoding a Nodamura virus (NoV) RNA2 genome segment having the sequence shown in SEQ ID No. 2; an isolated cDNA encoding a Pariacoto virus (PaV) RNA1 genome segment having the sequence shown in SEQ ID No. 3; and an isolated cDNA encoding a Pariacoto virus (PaV) RNA2 genome segment having the sequence shown in SEQ ID No. 4.
In another embodiment of the present invention, there is provided a nodavirus-based DNA expression vector comprising: a DNA-dependent RNA polymerase promoter; a cDNA encoding a nodaviral RNA-dependent RNA polymerase (RNA replicase) which is operably linked to the RNA polymerase promoter; a heterologous gene; nodaviral cis-elements which flank the RNA replicase cDNA and the heterologous gene; and a cDNA encoding a ribozyme that cleaves the transcripts of the expression vector, wherein said cleavag

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