HOW ESCHERICHIA-COLI CAN BIAS THE RESULTS OF MOLECULAR-CLONING - PREFERENTIAL SELECTION OF DEFECTIVE GENOMES OF HEPATITIS-C VIRUS DURING THE CLONING PROCEDURE

Cloned PCR products containing hepatitis C virus (HCV) genomic fragmen ts have been used for analyses of HCV genomic heterogeneity and protei n expression. These studies assume that the clones derived are represe ntative of the entire virus population and that subsets are not inadve rtently selected. The aim of the present study was to express HCV stru ctural proteins. However, we found that there was a strong cloning sel ection for defective genomes and that most clones generated initially mere incapable of expressing the HCV proteins. The HCV structural regi on (C-E1-E2-p7) was directly amplified by long reverse transcription-P CR from the plasma of an HCV-infected patient or from a control plasmi d containing a viable full-length cDNA of HCV derived from the same pa tient but cloned in a different vector. The PCR products were cloned i nto a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins. Twenty ra ndomly picked clones derived from the HCV-infected patient all contain ed nucleotide mutations leading to absence or truncation of the expect ed HCV products. Of 25 clones derived from the control plasmid, only 8 % were fully functional for polyprotein synthesis. The insertion of ex tra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number o f fully functional clones derived from the patient (42%) and from the control plasmid (72-92%). Nonrandom selection of clones during the clo ning procedure has enormous implications for the study of viral hetero geneity, because it can produce a false spectrum of genomic diversity. It can also be an impediment to the construction of infectious viral clones.