Latent baculovirus expression system

Details Latent baculovirus expression system Latent baculovirus expression system

1999-07-27

2001-06-12

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S235100, C435S455000, C435S456000, C435S091100, C435S091420, C435S091320

Reexamination Certificate

active

06245528

ABSTRACT:

BACKGROUND OF THE INVENTION
Viruses of the family Baculoviridae, commonly known as baculoviruses, are lethal pathogens of insects of the order Lepidoptera. These viruses typically kill their hosts by productive, lytic infection in insect tissues.
Baculoviruses generally contain a covalently closed, circular double-stranded DNA genome of about 90 to 160 kb in length. This genome is capable of accommodating relatively long heterologous sequences. In addition, a high level of structural viral protein synthesis is achieved during productive infection. Consequently, the baculovirus/insect cell system has become a popular protein expression vehicle for academic research and industrial applications. However, since high level expression is coincident with productive infection and late gene expression, the baculovirus expression system is limited by the eventual death of the baculovirus-infected culture.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a baculovirus containing a disruption in its endogenous p35 gene is able to establish a persistent infection (i.e., an infection in which the host are not lysed. Consequently, a persistent baculovirus expression culture system can be established by infecting any suitable host cell with a baculovirus having a disrupted p35 gene and an exogenous gene driven by a baculovirus early promoter (i.e., any promoter able to drive baculovirus expression in latent infection).
It was also discovered that cells harboring the p35-disrupted viruses are resistant to baculovirus super infection, i.e., infection by a second baculovirus. Thus, the p35-disrupted baculoviruses can be used to protect insects, especially commercially valuable insects (e.g., silkworms) from pathogenic baculoviruses.
Accordingly, the invention features a baculovirus including a disruption in its endogenous p35 gene. The baculovirus can further include a first sequence encoding a first RNA (e.g., a non-baculovirus RNA) and a baculovirus early gene promoter (e.g., an immediate early gene promoter) which drives expression of the first RNA. The first RNA can be a mRNA encoding a first protein (e.g., a non-baculovirus protein), such as a detectable protein (e.g., &bgr;galactosidase). The baculovirus can optionally also include a second sequence encoding a second non-baculovirus RNA. The second non-baculovirus RNA can be a mRNA encoding a second protein (e.g., a non-baculovirus protein), such as a selectable protein. A detectable protein is a protein whose expression is readily detectable, e.g., by fluorescent, luminescent, or chromogenic assays. A selectable protein is a protein whose expression confers a selectable phenotype on a cell. An example of a selectable phenotype is resistance to an antibiotic or antimitotic drug, such as neomycin or G418. Under some circumstances it can be useful to drive expression of the second protein from a baculovirus constitutive promoter, such as when the second protein confers drug resistance.
A baculovirus early promoter (e.g., a promoter derived from the baculovirus pagi gene) is a promoter, in the context of a baculovirus genome, that can drive expression in the early stage of the virus life cycle, i.e., just after viral entry and before expression of most viral structural proteins. A baculovirus constitutive promoter (e.g., a promoter derived from a heat shock protein-70 [hsp70] gene) is a promoter, in the context of a baculovirus genome, that can drive expression in all stages of the virus life cycle. A baculovirus early promoter or a baculovirus constitutive promoter is not necessarily derived from a baculovirus genome. Promoters derived from the genome of other organisms and viruses are useful in the baculovirus of the invention, as long as they satisfy the above constraints.
The invention also includes methods of expressing a non-baculovirus RNA or a non-baculovirus protein by introducing a baculovirus of the invention into a cell.
The invention also features a method of inhibiting baculovirus super infection in a cell by introducing into the cell a first baculovirus including a disruption in its endogenous p35 gene, and exposing the cell to a second baculovirus. By “exposing a cell to a second baculovirus” is meant placing the cell, or an organism containing the cell (e.g., a silkworm), in an environment suspected or known to contain a baculovirus. The environment can be artificial (e.g., a culture dish to which a baculovirus has been added) or in the wild (e.g., a forest where pathogenic baculoviruses are known to exist). As used herein, the term “inhibiting” includes both complete inhibition and partial inhibition.
A disruption of a gene is any deletion (e.g., deletion of a promoter sequence), insertion, or other mutation in the gene which renders the gene non-functional due to loss or misdirection of transcription, loss or misdirection of translation, or loss of an amino acid sequence necessary for protein function.
The baculoviruses and methods of the invention provide a new eukaryotic protein expression system useful in both academic and industrial settings. Unlike previous baculovirus expression systems, which are characterized by a self-limiting lytic infection, the expression systems made possible by the baculoviruses and methods of the invention utilize persistent, baculovirus-infected cultures. In addition, the baculoviruses described herein can be used to establish cells resistant to baculovirus super infection, a first step in protecting commercially valuable insects from baculovirus-induced disease.
Other features or advantages of the present invention will be apparent from the following drawings and detailed description, and also from the claims.


REFERENCES:
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Chao, Yu-Chan et al., “A 2.9-Kilobase Noncoding Nuclear RNA Functions in the Establishment of Persistent Hz-1 Viral Infection,” Journal of Virology, vol. 72, No. 3, p. 2233-2245, 1998.
Chao, Yu-Chan et al., “Differential Expression of Hz-1 Baculovirus Genes during Productive and Persistent Viral Infections,” Journal of Virology, vol. 66, No. 3, p. 1442-1448, 1992.
Clem, Rollie J. et al, “Prevention of Apoptosis by a Baculovirus Gene During Infection of Insect Cells,” Science, vol. 254, p. 1388-1390.
Clem, Rollie J. et al., “Influence of Infection Route on the Infectivity of Baculovirus Mutants Lacking the Apoptosis-Inhibiting Gene . . . ,” Journal of Virology, vol. 68, No. 10, p. 6759-6762, 1994.
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Hershberger, Pamela A. et al., “Site-Specific Mutagenesis of the 35-Kilodation Protein Gene Encoded byAutographa californica,Nuclear . . . ,” Journal of Virology, vol. 66, No. 9, p. 5525-5533, 1992.
Lee, Jin-Ching et al., “Superinfection-Induced Apoptosis and Its Correlation with the Reduction of Viral Progeny in Cells Persistently . . . ,” Journal of Virology, vol. 67, No. 12, p. 6989-6994, 1993.
Prikhod'ko, Elena A. et al., “Induction of Apoptosis by Baculovirus Transactivator IE1,” Journal of Virology, vol. 70, No. 10, p. 7116-7124, 1996.

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