title: Small interfering (Si) RNA mediated baculovirus replication reduction without affecting target gene expression.
abstract: The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant protein with posttranslational modification. Recombinant baculoviruses (such as Autographa californica multiple nuclear polyhedrosis virus) are especially useful in producing recombinant proteins and virus-like particles (VLPs) as biodrugs or candidate vaccines for the prevention of serious infectious diseases. However, during the bioprocessing of recombinant proteins in insect cells, baculovirus replication and viral budding are coincident. In some cases, residual baculovirus contaminants remain in the recombinant protein products, even though various purification processes are applied such as ion-exchange chromatography, ultracentrifugation, or gel filtration. To reduce unexpected contamination caused by replication and budding-out of the baculovirus, we designed short interfering (si) RNAs targeting glycoprotein 64 (GP64) or single-stranded DNA-binding protein (DBP) to inhibit baculovirus replication during overexpression of recombinant foreign genes. GP64 is known to be critical both for the entry of virions into cells and for the assembly of the budded virion at the cell surface. DBP is also essential for virus assembly by regulation of the capsid protein P39 and the polyhedrin protein. This study showed that GP64 expression was suppressed by GP64 siRNAs in Western blot experiments, while the expression of recombinant proteins was unaffected. In addition, transfection of GP64 siRNAs and DBP siRNAs reduced the level of baculovirus replication, compared with the treatment with scrambled siRNAs. However, DBP siRNA also suppressed the expression of recombinant proteins. In conclusion, our GP64 siRNAs showed that an interfering RNA system, such as siRNAs and short hairpin (sh) RNAs, can be applicable to reduce baculovirus contaminants during the bioprocessing of recombinant proteins in insect cells. Further investigation should be carried out to establish transformed insect cell lines with stable expression of corresponding interfering RNAs.
authors: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea.
authors: Han Saem
authors: HS
authors: Lee
authors: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea.
authors: Ho Yeon
authors: HY
authors: Lee
authors: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea.
authors: You-Jin
authors: YJ
authors: Kim
authors: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea.
authors: Hee-Dong
authors: HD
authors: Jung
authors: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea.
authors: Ki Ju
authors: KJ
authors: Choi
authors: Department of Life Science, Sogang University, Seoul 121-742, South Korea.
authors: Jai Myung
authors: JM
authors: Yang
authors: Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea.
authors: Sung Soon
authors: SS
authors: Kim
authors: Division of Influenza Virus, Center for Infectious Diseases, National Institute of Health, Korea CDC, 187 Osongsaemyong2-ro, 363-951 Cheongju-si, Chungbuk, South Korea. Electronic address: tigerkis@gmail.com.
authors: Kisoon
authors: K
authors: Kim
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism*; D016367:Baculoviridae / Q000254:growth & development / Q000502:physiology*; D001709:Biotechnology / Q000379:methods; D020868:Gene Silencing; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells; D018411:Spodoptera; D013678:Technology, Pharmaceutical / Q000379:methods; D019065:Virus Assembly / Q000187:drug effects*; D053586:Virus Internalization / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D034741:RNA, Small Interfering
keywords: Baculovirus; Glycoprotein 64; Single-stranded DNA binding protein; siRNA