Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase a. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.DNA polymerases are central enzymes in DNA replication. In prokaryotic systems, studies of DNA polymerases have been greatly aided by model systems to which both genetic and biochemical methods can be applied. The herpes simplex virus (HSV) DNA polymerase, which is essential for HSV replication (1), provides such a model system for the study of eukaryotic DNA polymerases. This enzyme resembles the mammalian replicative DNA polymerase a (pol a) in several respects, including nuclear localization (2) and sensitivities to inhibitors such as aphidicolin (3-7) and dideoxythymidine triphosphate (4). Recently, HSV DNA polymerase (HSV pol), DNA polymerase I of yeast, a variety of animal and bacterial virus DNA polymerases, and pol a were shown to share six regions of striking sequence similarity (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Of the viral polymerases sequenced to date, however, HSV pol appears most closely related to pol a (8). The six regions of sequence similarity have been designated regions I-VI by Wong et al. (8), with region I being most similar among the various polymerases and region VI being the least similar (Fig. 1). Such sequence similarities suggest conservation during evolution and important functional roles for these regions.Despite these similarities, HSV pol is more sensitive than pol a to a number of inhibitors and thus serves as the ultimate target for a number of selective antiviral drugs. These include the pyrophosphate (PP,) analogs phosphonoacetic acid (PAA) and phosphonoformic acid (20, 21), and the triphosphates of nucleoside analogs such as acyclovir (ACV), vidarabine, ganciclovir, and bromovinyldeoxyuridine (BVdU) (22-25). In addition, aphidicolin appears to inhibit HSV pol competitively with certain deoxynucleoside triphosphates (dNTPs) (5-7).The availability of viral pol mutants that exhibit altered se...

The herpes simplex virus DNA polymerase provides an excellent model for studies of eukaryotic replicative polymerases. We report here the nucleotide sequence of the gene which encodes this enzyme. The gene includes a 3705-base-pair major open reading frame capable of encoding a Mr 136,519 polypeptide, in rough agreement with previous estimates of the size of the major polypeptide found in partially purified viral polymerase preparations. The predicted polymerase polypeptide shares extensive sequence homology with the Epstein-Barr virus open frame predicted to encode DNA polymerase and with a 13-amino acid segment of adenovirus 2 DNA polymerase. Mutations conferring altered sensitivity to antiviral deoxynucleoside triphosphate analogs, pyrophosphate analogs, or aphidicolin from eight different mutants map within the region encoding the carboxyl-terminal portion of the predicted polymerase polypeptide. Two of these are separated by a distance corresponding to at least 228 amino acids. We propose that this region of the gene encodes a polypeptide domain that contains the binding sites for deoxynucleoside triphosphates and pyrophosphate.

We have previously shown targeting of DNA to hepatocytes using an asialoorosomucoid-polylysine (AsOR-PL) carrier system. The AsOR-PL conjugate condenses DNA and facilitates entry via specific receptor-ligand interactions. In these studies, our objective was to determine if AsOR-PL conjugates protect bound DNA from nuclease attack. Double-stranded plasmid or single-stranded oligonucleotide DNA, alone or bound to conjugate, was incubated under conditions mimicking those encountered during in vitro and in vivo transfections. The results showed that complexed DNA was effectively protected from degradation by serum nucleases. Degradation of single-stranded oligonucleotides was inhibited 3- to 6-fold in serum during 5 hours of incubation. For complexed plasmids, greater than 90% remained full-length during 1.5 and 3 hour incubations in serum or culture medium containing 10% serum, respectively. Uncomplexed plasmid was completely degraded after 15 minutes in serum or 60 minutes in medium. In cell lysates, the conjugate was not effective in inhibiting endonuclease activity; plasmids were readily converted from supercoiled to open circular and linear forms. However, the resultant nicked forms were substantially protected from further degradation during one hour of incubation compared to plasmid alone. Under all conditions complexed DNA did not readily dissociate from the conjugate. Overall, for both single and double-stranded DNA, AsOR-PL conjugates conferred substantial protection from nuclease degradation.

In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.

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The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.

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Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian cells. Accelerating activity in biotherapeutic drug development, demand for protein-based reagents, vaccine research, and large initiatives in structural and functional studies of proteins have propelled the need to generate moderate to high amounts of recombinant proteins and other macromolecules in a flexible and rapid manner. Progress over the last 10-15 years has demonstrated that transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. This review will summarize developments in this field, properties of the components of a transient expression system that enable maximal protein production, and detailed protocols for this application.