David Rowlands collection

Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a ‘minigenome’ derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but β-galactosidase activity was limited to 10–20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis δ virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis δ virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host–cell interactions.

Foot-and-mouth disease virus (FMDV) attached to pig kidney cells at 0 °C and could only be recovered in a form with a sedimentation coefficient and buoyant density lower than that of the native virus. Incubation of the virus-cell complex at 37 °C caused disruption of about 80% of the particles into a 12S protein sub-unit that had the same polypeptide composition as that produced by reducing the pH of the virus below pH 7. The remaining 20% had the same polypeptide and RNA composition as the native virus but it had a lower sedimentation coefficient, buoyant density and specific infectivity. These lower values are probably due to the association of the virus with cell membrane components. The 12S subunits were shown to be located inside the cell, indicating that disruption of the virus had occurred within the cell. The results are discussed in relation to the different cell mediated alteration of other picornaviruses.

Unfractionated harvests of foot-and-mouth disease virus grown in baby hamster kidney cells fixed complement with both heterotypic and homotypic antisera but the freshly prepared intact virus (25 nm. component) from these harvests fixed complement only with the homotypic antiserum. Storage at 4° or heating at 37° released an antigen from the 25 nm. component which fixed complement with heterotypic serum. This antigen could also be prepared by mixing the 25 nm. component with baby hamster kidney cells but it was obtained in greatest yield by disrupting with guanidine. It had a sedimentation coefficient of 14S in sucrose gradients. Serum from hyperimmunized infected guinea pigs which had been absorbed with excess homotypic 25 nm. component fixed complement with the disrupted virus but not with intact virus. The disrupted virus also reacted with heterotypic antiserum produced by inoculation of guinea pigs with inactivated 25 nm. component, providing further evidence that the antigen is a structural component of the virus particle.