Phosphonoformic Acid Inhibits Viral Replication by Trapping the Closed Form of the DNA Polymerase

Abstract

Phosphonoformic acid (PFA, foscarnet) belongs to a class of antiviral drugs that inhibit the human cytomegalovirus DNA polymerase (UL54) by mimicking the pyrophosphate leaving group of the nucleotide transfer reaction. Difficulties expressing UL54 have hampered investigation of the precise structural requirements rendering inhibition by this drug. However, a previously engineered chimeric DNA polymerase, constructed by mutating the homologous polymerase from bacteriophage RB69 (gp43) to express several variable elements from UL54, can bypass this obstacle because of its favorable expression and acquired sensitivity to PFA (Tchesnokov, E. P., Obikhod, A., Schinazi, R. F., and Götte, M. (2008) J. Biol. Chem. 283, 34218 -34228). Here, we compare two crystal structures that depict the chimeric DNA polymerase with and without PFA bound. PFA is visualized for the first time in the active site of a DNA polymerase, where interactions are resolved between the PP i mimic and two basic residues absolutely conserved in the fingers domain of family B polymerases. PFA also chelates metal ion B, the cation that contacts the triphosphate tail of the incoming nucleotide. These DNA complexes utilize a primer-template pair enzymatically chain-terminated by incorporation of acyclo-GMP, the phosphorylated form of the anti-herpes drug acyclovir. We postulate that the V478W mutation present in the chimera is critical in that it pushes the fingers domain to more readily adopt the closed conformation whether or not the drug is bound. The closed state of the fingers domain traps the variant polymerase in the untranslocated state and increases affinity for PFA. This finding provides a model for the mechanism of UL54 stalling by PFA. The human cytomegalovirus (HCMV)3 belongs to the herpesviridae family, into which several important human pathogens are classified. A primary infection of this virus in pregnant mothers can be devastating to the developing fetus, resulting in several congenital disorders. Although a substantial portion of the global adult population is currently HCMV-seropositive, a functional immune system can hold the virus in check. For immunocompromised individuals, on the other hand, infection with HCMV presents a severe threat (1-3). Accordingly, antiviral therapy is used as both prophylaxis and therapeutic strategies in solid organ transplant recipients, who receive concomitant immunosuppressants. In this setting, infection with the virus is a major cause of morbidity and mortality.The limited pharmacological options available for treatment of HCMV largely consist of nucleoside analog inhibitors that target the gene product of UL54, a family B DNA polymerase. Although some inhibitors, such as cidofovir, are chemically synthesized as acyclic nucleoside phosphonates, most nucleoside analogs require monophosphorylation by a viral kinase, such as UL97 in HCMV, before other cellular kinases convert these drugs into triphosphorylated substrates of the DNA polymerase (4). Following incorporation into the viral DNA, these non-n...

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

“…The Hε-dependent and -independent protein priming assays we have now developed should facilitate the dissection of this important early conformational change that HP undergoes during protein-primed DNA synthesis and help efforts to target this for developing novel HP inhibitors for antiviral therapy. Im-portantly, the utility of PFA as a sensitive detector of polymerase conformations was verified by recent structural studies using a chimeric DNA polymerase derived from a cytomegalovirus (73).…”

Direct Observation of Translocation in Individual DNA Polymerase Complexes

“…If phi29 DNAP binary complexes are in equilibrium between the fingers-open and fingers-closed states, as shown by smFRET for the A-family polymerase KF (27), this steric block may be relieved in a closed binary complex, permitting the movement to the pre-translocation state. Indeed, a candidate for such a closed binary complex, captured in the pre-translocation state, was observed in a recent structure of a chimeric RB69-UL54 B-family DNAP (28). The amplitude fluctuations observed in complexes atop the pore may thus reflect an equilibrium between the fingers-open and fingers-closed states inherent in phi29 DNAP-DNA binary complexes.…”

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

“…The pyrophosphate analog phosphonoformic acid (PFA or foscarnet) was shown to inhibit RT by trapping the enzyme in the pre-translocational state (12,13). The observed preference of PFA for the pre-translocational form of the polymerase⅐DNA complex was recently validated by the first crystal structure of PFA bound to a DNA polymerase, which showed PFA binding and stabilization of the closed enzyme conformation leading to the formation of an untranslocated form of the polymerase⅐DNA complex (14). In contrast, the more recently discovered scaffold of indolopyridones (INDOPY-1) (15,16) traps RT in the post-translocational state (15).…”