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PubMed1988

Co-expression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors.

The hepatitis B (HB) virus DNA sequences coding for the pre-core (preC) or C antigens (HBpcAg, HBcAg) have been inserted into the baculovirus plasmid transfer vector, pAcYM1, such that the HB viral sequences are under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with either of the derived recombinant plasmids in the presence of infectious AcNPV DNA yielded recombinant, polyhedrin-negative viruses that expressed high levels of the respective HBpcAg or HBcAg (representing approx. 5 to 10% and approx. 40% of the stained cellular proteins, respectively). The particulate 27 nm HBcAgs have been purified to homogeneity from infected cell extracts by density gradient centrifugation. Dual expression transfer vectors containing the HBcAg gene sequences and the coding sequences of the HB viral S antigen (HBsAg), each gene under the control of its own copy of the polyhedrin promoter, have also been constructed and used to derive recombinant viruses. The recombinant with the HB C and S genes expressed high levels of the HBcAg (approx. 40% of the cellular proteins) and low levels of the HBsAg (approx. 2% of the stained cellular proteins). Dual expression, occluded, recombinant baculoviruses that make HBsAg, as well as the AcNPV polyhedrin protein, have been prepared that are highly infectious for Trichoplusia ni caterpillars, allowing reproducible preparation of the antigen in larvae. Using radioimmunoassays (RIAs) and ELISAs, the recombinant HBcAg (RIA) and HBsAg (ELISA) have been used to identify human antibodies to HB virus with results that compare favourably with the data obtained with non-recombinant antigens.

Amino Acid Sequence↗
PubMed2002

Significant increase in recombinant protein production of a virus-infected Sf-9 insect cell culture of low MOI under low dissolved oxygen conditions.

Spodoptera frugiperda Sf-9 insect cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (beta-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the cell growth following viral infection was greatest and beta-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4-8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 cells. These results indicated that for a baculovirus-infected Sf-9 insect cell culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intracellularly only at 5% DO, coinciding with beta-galactosidase production.

Journal Article↗
PubMed1999

Identification of inhibitory mutants of Cauliflower mosaic virus movement protein function after expression in insect cells.

Cauliflower mosaic virus (CaMV) encodes a movement protein (MP) which forms tubules in vivo and mediates the translocation of virus particles through plasmodesmata. The relationship between CaMV MP structure and function, in isolation from the complete virus infection, was studied by using MP expression in insect cells. The study allowed the MP domains necessary for tubule formation to be identified and potential MP-MP interactions to be investigated by using double infections with recombinant baculoviruses. Two MP domains which interfered with the ability of the wild-type MP to form tubules were identified. These mutant domains appeared to act as competitive, rather than dominant negative, inhibitors.

Animals↗
PubMed2005

Enhancement of cauliflower mosaic virus 35S promoter in insect cells infected with baculovirus.

We happened to discover that the cauliflower mosaic virus (CaMV) 35S promoter inserted into a recombinant Autographa californica multicapsid nucleopolyhedrovirus (rAcMNPV) was strongly activated during the replication of the recombinant virus in Spodoptera frugiperda (Sf9) cells. The expression of the luciferase gene from the 35S promoter in rAcMNPV was remarkably increased late in infection and was resistant to alpha-amanitin treatment. Primer extension indicated that transcriptional initiation from the 35S promoter in Sf9 cells occurred within one of the two baculoviral late promoter TAAG motifs located in the vicinity of the transcription start site in plant cells. These observations suggested that the CaMV 35S promoter served as a transcription start site for AcMNPV-induced RNA polymerase.

Animals↗
PubMed1987

Secretion of particles of hepatitis B surface antigen from insect cells using a baculovirus vector.

The coding sequences of the hepatitis B virus surface antigen were inserted into a baculovirus transfer vector produced from Autographa californica nuclear polyhedrosis virus (AcNPV) so that the foreign gene was under the control of the AcNPV polyhedrin promoter. Spodoptera frugiperda cells infected with the derived recombinant baculovirus produced and secreted 22 nm particles containing the hepatitis B surface antigen. The particles had morphological and antigenic properties identical to those of 22 nm particles isolated from the plasma of chronic active hepatitis patients.

Animals↗
PubMed1988

Evidence for phosphorylation of trans-activator p40x of human T-cell leukemia virus type I produced in insect cells with a baculovirus expression vector.

Human T-cell leukemia virus type I (HTLV-I) encodes a trans-activator protein p40x which positively regulates transcription of the viral RNA as well as interleukin-2 and its receptor genes. We placed a cDNA coding for p40x in baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) expression vectors. Infection of BmN cells derived from an insect, B. mori (silkworm), with a recombinant virus led to the production of soluble p40x. The biological activity of the recombinant p40x was demonstrated by introducing the protein into intact NIH 3T3 cells that had been selected for genomic integration of HTLV-I LTR connected with the CAT gene. Immunocytochemical and cell fractionation analyses showed the localization of p40x in both the cytoplasmic and nuclear fractions of BmN cells. Analyses of 32P-labeled proteins of BmN cells by cell fractionation and subsequent immunoprecipitation revealed that the p40x present in each subcellular fraction was phosphorylated. The post-translational modification was inhibited by the addition of a protein kinase inhibitor K252a during the metabolic labeling of BmN cells. Phosphoamino acid analysis indicated that the phosphorylation occurred on serine residues of p40x.

Animals↗
PubMed2004

Membrane binding properties and terminal residues of the mature hepatitis C virus capsid protein in insect cells.

The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with alpha-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.

Amino Acid Sequence↗
PubMed1992

Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells.

The three capsid proteins VP1, VP2, and VP3 of the adeno-associated virus type 2 (AAV-2) are encoded by overlapping sequences of the same open reading frame. Separate expression of these proteins by recombinant baculoviruses in insect cells was achieved by mutation of the internal translation initiation codons. Coexpression of VP1 and VP2, VP2 and VP3, and all three capsid proteins and the expression of VP2 alone in Sf9 cells resulted in the production of viruslike particles resembling empty capsids generated during infection of HeLa cells with AAV-2 and adenovirus. These results suggest a requirement for VP2 in the formation of empty capsids. Individual expression of the AAV capsid proteins in HeLa cells showed that VP1 and VP2 accumulate in the cell nucleus and VP3 is distributed between nucleus and cytoplasm. Coexpression of VP3 with the other structural proteins also led to nuclear localization of VP3, indicating that the formation of a complex with VP1 or VP2 is required for accumulation of VP3 in the nucleus.

Animals↗
PubMed1995

Immunogenicity of influenza A virus N2 neuraminidase produced in insect larvae by baculovirus recombinants.

Influenza A virus neuraminidase (NA) from A/Udorn/72 (H3N2) was expressed by recombinant baculovirus-infected insects. The recombinant NA was enzymatically active. Enzyme activity was neutralized by polyclonal antisera raised against virion-extracted NA. NA produced in whole insects by a baculovirus expression system is antigenically indistinguishable from virion NA by polyclonal antisera in functional assays (NI) and in ELISA, and is highly immunogenic without adjuvant. It is equivalent in immunogenicity to NA purified from influenza virus. Our results indicate that baculovirus-produced NA could be used as a source for large quantities of purified N2-NA for vaccine use.

Animals↗
PubMedDate not supplied

[Expression of hepatitis A virus procapsids in the insect cells infected by recombinant baculovirus].

The recombinant baculocvirus containing genome P1-2A-P3 of hepatitis A virus (HAV) was constructed and used for infecting the Sf9 insect cells. It was demonstrated that the deletion of 2BC from HAV polyprotein and the insertion of a new 3C protease cleavage site between P1-2A and P3 did not interfere with the processing of polyprotein or with forming the 70S-procapsids. The identity of the protein contents as well as of morphological and antigen characteristics, obtained in Sf9-cells, to HAV empty capsids, which take shape in the infected mammal cells, proves that it is possible to use them in making the vaccine and diagnostic preparations.

Animals↗