Establishment of stable HeLa cell lines expressing enzymatically active hepatitis C virus RNA polymerase.

The hepatitis C virus RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target for antiviral drug development. In this study, stable HeLa cell lines with an integrated NS5B gene were selected by G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated NS5B genes were demonstrated by RT-PCR and Western blot analysis. Further analysis demonstrated enzymatic activity of the expressed NS5B polymerase. The stable HeLa cell lines should be useful for the identification of NS5B inhibitors and for studying the mechanisms of HCV replication.

In vitro expression of the human cytomegalovirus DNA polymerase gene: effects of sequence alterations on enzyme activity.

Genomic DNA of the Towne strain human cytomegalovirus polymerase (pol) gene (4.4-kb RsrII-NcoI segment of the EcoRI J fragment) was cloned into plasmids containing either the T3 or the T7 promoter for in vitro transcription-translation studies. The translation efficiency of unmodified pol cRNA was poor in this system and could not be improved by capping. However, the efficiency could be enhanced by replacing the leader sequence with a 40-bp AT-rich sequence derived from an alfalfa mosaic virus, R4. pol cRNA directed the synthesis of a 140-kDa polypeptide in a rabbit reticulocyte translation system. The in vitro-translated wild-type enzyme possessed significant polymerization activity which could be stimulated by salt as could that of the authentic enzyme purified from virus-infected cells. To study the critical domains of this enzyme, nine mutations were introduced into the pol gene around the conserved domains of eukaryotic polymerase by oligonucleotide-directed mutagenesis. Two constructs with mutations at amino acid residues 323 to 325 (M32QS) and 725 to 726 (M72II) remained active, with partial loss of enzyme activity, while the enzyme activities of other mutants with alterations at four domains located around amino acid residues 729 to 730 (M73HN), 804 to 807 (M80 and DE80), 910 to 913 (M91 and DE91), and 962 to 964 (M96 and DE96) were abolished. DNA template and triphosphate binding assays indicated that the mutation at 804 to 807 (conserved region III) lost the ability to bind DNA template, and four mutants, M73HN (within conserved region II), M80 (in region III), M91 (in region I), and M96 (around region V [962 to 964; amino acid sequence KKR]), failed to bind deoxyribonucleoside triphosphate. These data suggest that conserved region III is essential for DNA template binding, while residues between conserved region II and V (725 to 964) are involved in triphosphate binding.

A point mutation in the polyhedrin gene of a baculovirus, Autographa californica MNPV, prevents crystallization of occlusion bodies.

The polyhedrin gene region of the Autographa californica nuclear polyhedrosis virus(AcMNPV) morphology mutant M29 has been characterized by genetic and physical techniques. Recombination analysis of mutants M29 and AcM5polyl demonstrated that wild-type polyhedrin recombinants could be obtained and that the DNA restriction patterns of the recombinant viruses were identical to wild-type AcMNPV DNA. Marker rescue experiments using the wild-type AcMNPV EcoRI I fragment indicated that the morphology mutation responsible for the M29 phenotype was located in the 0.0 to 5.9 % region of the genome. Direct DNA sequencing of the BamHIF fragment from M29 showed a single point mutation at position 253 of the polyhedrin gene. This mutation caused a substitution of phenylalanine for leucine at amino acid 84 of the M29 polyhedrin protein. These results indicated the necessity of amino acid conservation in the polyhedrin amino acid sequence for proper folding and assembly of the polypeptide into occlusion bodies.