Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

Abstract

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2-to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by viral DNA polymerases (Pols) can result in the selective loss of viral replicative activity in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells, which contain high levels of dNTPs (10,13,16,17). Actively dividing tumor cells can contain between 10-and 30-times-higher dNTP concentrations than primary cells (16), providing a biochemical basis on which it might be possible to develop viral vectors that can selectively replicate in tumor cells but not normal cells. Since adenovirus vectors have been well studied as candidate oncolytic agents (8,9,11,14,19), we decided to focus our analysis on this virus and to determine the feasibility of generating modified adenoviruses containing mutated DNA polymerases with reduced dNTP binding affinities.The adenovirus DNA polymerase (AdPol) has been identified as a 140-kDa DNA polymerase of the alpha-like, Pol B family of polymerases (6, 12). Such polymerases contain several conserved motifs that are essential for polymerase function and contain amino acid residues that are necessary for dNTP binding (2, 29, 33), template DNA binding (4, 7, 15), and polymerase activity (5,23,30). ...

Intracellular nucleotide levels and the control of retroviral infections

“…Furthermore, based on these biochemical and virological findings (15, 29, 61, 68), it was hypothesized that other viruses containing DNA polymerase mutants with reduced dNTP binding affinity may replicate exclusively in cell types harboring elevated dNTP pools such as cancer cells. This was confirmed by a series of adenovirus polymerase variants that replicate preferentially in cancer cells and also function as oncolytic viruses (10). …”

Ganciclovir Inhibits Human Adenovirus Replication and Pathogenicity in Permissive Immunosuppressed Syrian Hamsters

“…The Ad5 genomeencoded DNA polymerase (Ad5 Pol) with a six-histidine affinity tag at the C terminus was expressed from a baculovirus/Ad5 Pol vector in SF9 cells (41). The Ad5 Pol was purified to near homogeneity (41).…”

“…The Ad5 genomeencoded DNA polymerase (Ad5 Pol) with a six-histidine affinity tag at the C terminus was expressed from a baculovirus/Ad5 Pol vector in SF9 cells (41). The Ad5 Pol was purified to near homogeneity (41). Polymerase extension was tested on a substrate containing a 5= 32 P-labeled 20-mer annealed to a template 30-mer as described previously (41,42).…”

“…The Ad5 Pol was purified to near homogeneity (41). Polymerase extension was tested on a substrate containing a 5= 32 P-labeled 20-mer annealed to a template 30-mer as described previously (41,42). The reactions were set up in a 20-l reaction volume using 0.25 nM radiolabeled substrate in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 1 mg/ml bovine serum albumin (BSA), 5 mM MgCl 2 , 1 mM dithiothreitol (DTT), 60 M deoxynucleoside triphosphates (dNTPs), and 5% glycerol.…”

Cidofovir Diphosphate Inhibits Adenovirus 5 DNA Polymerase via both Nonobligate Chain Termination and Direct Inhibition, and Polymerase Mutations Confer Cidofovir Resistance on Intact Virus

“…Production and purification of AdV5 Pol from baculovirus. The Baculovirus transfer vector pAcGP67A-AdPol6xHis containing the AdV5 viral Pol region was generated previously (23). Gene segments containing the T87I (GeneArt) and V303I mutations (IDT) were digested with BamHI-SbfI and SbfI-BlpI endonucleases, respectively, and subsequently cloned into the pAcGP67A-AdPol6xHis vector.…”