Baculovirus expression system

The present invention relates to novel baculoviruses, baculovirus vectors, transfer vectors and to host cells comprising these viruses or vectors. Furthermore, the present invention relates to methods for producing the novel baculoviruses and baculovirus vectors. The present invention also pertains to a method for producing a protein of interest using the novel baculoviruses and baculovirus vectors. Baculovirus expression system Field of the invention The present invention relates to modified baculoviruses comprising in the genome an inactive form of a nucleotide sequence, said nucleotide sequence being enriched in defective interfering virus particles of the baculoviruses. Furthermore, the invention pertains to a method for preparing these baculoviruses and a method for producing a protein of interest using such baculoviruses. Background of the invention Baculoviruses are large enveloped, circular dsDNA viruses that can infect over 600 different types of invertebrates, but preferably infect insects. They are widely used as bio-insecticides in agriculture and forestry and can be genetically engineered to improve their effectiveness. More recently, baculoviruses were shown to have potential as gene delivery vectors for gene therapy or as vectors for surface display of complex eukaryotic proteins. Furthermore, baculoviruses are particularly well-suited for use as eukaryotic cloning and expression vector. Advantages of the baculovirus expression sytem are among others that the expressed proteins are almost often soluble, correctly folded and biologically active. Further advantages include high protein expression levels, faster production, suitability for expression of large proteins and suitability for large-scale production. In the large-scale production of baculoviruses as bio-insecticides or in large-scale or continuous production of heterologous proteins using the baculovirus expression system in insect cell bioreactors, the passage effect is a major obstacle. This effect is notable as a significant drop in production by prolonged virus passaging in insect cell culture and is a result of the accumulation of defective interfering particles (DIs). These DIs are rapidly generated in cell culture and become predominant after prolonged passaging, meanwhile interfering with the replication of intact helper virus and the production of polyhedra. It is known that DIs have retained cis-acting elements important for baculovirus DNA synthesis, but what sequences, if any, are involved in the generation of DIs is still enigmatic. Surprisingly, we have found that inactivating a nucleotide sequence present in the genome of baculovirus, said nucleotide sequence being enriched in defective interfering virus particles, leads to enhanced stability of virus and polyhedra production and therefore prevents the negative consequences of the above-mentioned passage effect. Description of the invention Definitions Herebelow follow definitions of terms as used in the present invention. Homologous The term "homologous" is used herein to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell and is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically be operably linked to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. Additionally, the term "homologous" is also used herein in the context of amino acid sequences is intended to encompass both amino acid identity and similarity between the two sequences. To determine the percent homology of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for optimal alignment with the other protein). The amino acid residues at corresponding amino acid positions are then compared and when a position in one sequence is occupied by the same or a similar amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position. The percent homology between two sequences, therefore, is a function of the number of identical or similar positions shared by two sequences. Computer algorithms known in the art can be used to optimally align the two amino acid sequences to be compared and to define similar amino acid residues. Preferably, the Basic Local Alignment Search Tool (BLAST) algorithm is used to compare the two amino acid sequences to thereby determine the percent homology between the two sequences. The term "homologous" as used in the context of nucleotide sequences is intended to refer to nucleotide sequence identity between the two sequences. To determine the percent homology of two nucleotide sequences, the sequences are aligned for optimal comparison purposes. The nucleic acid bases at corresponding nucleotide positions are then compared and when a position in one sequence is occupied by the same nucleic acid base as the corresponding position in the other sequence, then the molecules are homologous at that position. The percent homology between two sequences, therefore, is a function of the number of identical positions shared by two sequences. The Basic Local Alignment Search Tool (BLAST) algorithm mentioned above can also be used to compare the two nucleotide sequences to thereby determine the percent homology between the two sequences.