In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2.

The aim of this study was to describe the generation of a PCV2 (porcine circovirus type 2) infectious clone (pIC-PCV2) and its infectivity under in vitro and in vivo conditions. The constructed pIC-PCV2 contained the whole PCV2 genome from a German isolate together with a partial duplication of 467 bp. PK-15 cells were transfected with pIC-PCV2 and an indirect immune fluorescence assay (IFA) was performed 7 days post-transfection. The PCV2 Cap gene was expressed in approximately 20 % of the cultured cells, and only the recombination product, and not pIC-PCV2, was subsequently detected by PCR and Southern blot. This result indicated that infection by pIC-PCV2 delivered genomic PCV2 DNA specifically into susceptible cells and led to the expression of a functional virus genome. Eighteen 30- to 40-day-old conventional pigs were distributed into three groups. Group 1 pigs (n=6) were inoculated intranasally (i.n.) with a Spanish isolate of PCV2 propagated in cell culture; pigs from group 2 (n=6) were inoculated with pIC-PCV2 intramuscularly (i.m.), and the last group of pigs (n=6) was inoculated with pIC-PCV2 intraperitoneally (i.p.). All pigs remained clinically healthy during the whole experimental period (35 days). Pigs that received pIC-PCV2 i.p. and i.m., as well as those PCV2 i.n. inoculated, became infected based on an in situ hybridization (ISH), PCR, TaqMan PCR and serological results. The results of this study confirm that cloned PCV2 genomic DNA is infectious both in vitro and in vivo, and is able to cause PMWS-like lesions in i.p. and i.m. experimentally inoculated pigs.

Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway.

The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by lon-deficient Bl21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.

The expression of recombinant genes from bacteriophage lambda strong promoters triggers the SOS response in Escherichia coli.

The production of several non-related heterologous proteins in recombinant Escherichia coli cells promotes a significant transcription of recA and sfiA SOS DNA repair genes. The activation of the SOS system occurs when the expression of plasmid-encoded genes is directed by the strong lambda lytic promoters, but not by IPTG-controlled promoters either at 37 or at 42 degrees C, and it is linked to an extensive degradation of the proteins after their synthesis. The triggering signal for the SOS response could be an important arrest of cell DNA replication observed within the first hour after the induction of recombinant gene expression. The stimulation of this DNA repair system can partially account for the toxicity exhibited by recombinant proteins on actively producing E. coli cells.

Antigenicity of VP60 structural protein of rabbit haemorrhagic disease virus.

Five overlapping segments of the VP60 capsid protein gene of rabbit haemorrhagic disease virus have been expressed in E. coli under the control of the T7 RNA polymerase. After purification, the antigenicity of these denatured protein segments has been studied by reactivity with sera from both naturally infected and vaccinated animals in Western blot analysis. The amino terminus segments of the protein (comprising the first 175 amino acids) are highly reactive with the tested sera, between 10 and 100 fold more than any of the segments reproducing the carboxy half of VP60, which is believed to be solvent-exposed in the virus particles. These results strongly suggest that the antigenic structure of the carboxy moiety of VP60 is mainly based on conformation-dependent B-cell epitopes whereas the amino terminal region of VP60 contains continuous antigenic determinants for the immune response elicited during both virus infection and exposure to the inactivated vaccine.

Reversible activation of a cryptic cleavage site within E. coli beta-galactosidase in beta-galactosidase fusion proteins.

The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.

Polylinker-encoded peptides can confer toxicity to recombinant proteins produced in Escherichia coli.

Three DNA segments encoding fragments of the p60 capsid protein of rabbit haemorrhagic disease virus (RHDV) have been cloned and expressed in Escherichia coli. The cDNAs were placed under the control of the T7 promoter in a pET-derived expression vector designed to produce C-terminal histidine tail fusions. By using different cloning strategies, cell toxicity exhibited by some of the expressed products was dramatically affected, coincident with minor modifications in the carboxy terminus sequences of the recombinant proteins. In particular, the presence of a nonhydrophobic peptide encoded by polylinker sequences promotes cell death and a reduced yield after induction of gene expression, whereas a histidine tail has no detectable effect. These data point out the critical role that needless peptide tails, accidentally introduced into recombinant proteins by nonrelevant DNA stretches, can have on protein expression and final yield of a production process.

The position of the heterologous domain can influence the solubility and proteolysis of beta-galactosidase fusion proteins in E. coli.

The VP1 protein (23 kDa) of the foot-and-mouth disease virus has been produced in MC1061 and BL21 E. coli strains as beta-galactosidase fusion proteins, joined to either the amino and/or the carboxy termini of the bacterial enzyme. In BL21, devoid of La protease, all the recombinant fusion proteins are produced at higher yields than in MC1061, and occur mainly as inclusion bodies. The fusion of VP1 at the carboxy terminus yields a protease-sensitive protein whose degradation releases a stable, enzymatically active polypeptide indistinguishable from the native beta-galactosidase. On the contrary, when the same viral domain is fused to the amino terminus, the resulting chimeric protein is resistant to proteolysis even in the soluble form. These data demonstrate that the position of the heterologous domain in beta-galactosidase fusion proteins would not be irrelevant since it can dramatically influence properties of biotechnological interest such as solubility and proteolytic resistance.

Microheterogeneity of p60 capsid protein and the encoding gene among contemporary isolates of rabbit hemorrhagic disease virus.

The gene encoding the p60 capsid protein from a Spanish isolate of the rabbit hemorrhagic disease virus (RHDV) has been cloned and sequenced. Both cDNA and the derived protein sequences have been compared with those from several contemporary European RHDV isolates. Genetic heterogeneity among co-circulating viruses is nearly constant along the p60-encoding gene, around 0.034 substitutions per nucleotide, and it does not allow prediction of the preferential regions for long-term fixation of mutations. However, sequence comparisons with the more distant, but phylogenetically closely related calicivirus, the European brown hare syndrome virus (EBHSV), reveal a great extent of genetic variability within both a segment of p60 gene (nucleotides 900-1300) and the encoded polypeptide, suggesting the existence of strong selective pressures acting over this region of the capsid protein.

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli.

The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype C1 virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.

Fine regulation of cI857-controlled gene expression in continuous culture of recombinant Escherichia coli by temperature.

The expression at different temperatures of the lacZ gene, which is controlled by the lambda pL and pR tandem promoters and the cI857 temperature-sensitive repressor, was studied in Escherichia coli continuous cultures. At temperatures between 30 and 42 degrees C, beta-galactosidase activity behaved according to an exponential equation. By inducing a culture at a temperature within this range, predefined, nearly constant submaximal levels of gene expression and recombinant product yield can be obtained.