The processivity factor complex of feline herpes virus-1 is a new drug target.

Feline herpes virus-1 (FHV-1) is ubiquitous in the cat population and is a major cause of blindness for which antiviral drugs, including acyclovir, are not completely effective. Recurrent infections, due to reactivation of latent FHV-1 residing in the trigeminal ganglia, can lead to epithelial keratitis and stromal keratitis and eventually loss of sight. This has prompted the medical need for an antiviral drug that will specifically inhibit FHV-1 infection. A new antiviral target is the DNA polymerase and its associated processivity factor, which forms a complex that is essential for extended DNA strand synthesis. In this study we have cloned and expressed the FHV-1 DNA polymerase (f-UL30) and processivity factor (f-UL42) and demonstrated that both proteins are required to completely synthesize the 7249 nucleotide full-length DNA from the M13 primed-DNA template in vitro. Significantly, a known inhibitor of human herpes simplex virus-1 (HSV-1) processivity complex was shown to inhibit FHV-1 processive DNA synthesis in vitro and block infection of cells. This validates using f-UL42/f-UL30 as a new antiviral drug target to treat feline ocular herpes infection.

Anthrax edema factor potency depends on mode of cell entry.

Anthrax edema factor (EF) is a highly active calmodulin-dependent adenylyl cyclase toxin that can potently raise intracellular cAMP levels causing a broad range of tissue damage. EF needs anthrax protective antigen (PA) to enter into the host cell and together they form edema toxin. Here, we examine factors that are critical for edema toxin cell entry and potency. In Y1, 293T and mouse embryonic fibroblast cells, EF causes cell rounding, aggregation, and sometimes detachment via protein kinase A but not Epac. The rate-limiting step for these EF-mediated effects is cellular entry via the anthrax toxin receptor. Finally, EF potency is also enhanced if the EF adenylyl cyclase domain is transfected into host cells, even in the absence of the anthrax PA-binding domain. These results indicate that the effects of EF in cells can differ dependent upon the mode of cellular entry of the adenylyl cyclase.

Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor.

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM and uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.

Structural and kinetic analyses of the interaction of anthrax adenylyl cyclase toxin with reaction products cAMP and pyrophosphate.

Anthrax edema factor (EF) raises host intracellular cAMP to pathological levels through a calcium-calmodulin (CaM)-dependent adenylyl cyclase activity. Here we report the structure of EF.CaM in complex with its reaction products, cAMP and PP(i). Mutational analysis confirmed the interaction of EF with cAMP and PP(i) as depicted in the structural model. While both cAMP and PP(i) have access to solvent channels to exit independently, PP(i) is likely released first. EF can synthesize ATP from cAMP and PP(i), and the estimated rate constants of this reaction at two physiologically relevant calcium concentrations were similar to those of adenylyl cyclase activity of EF. Comparison of the conformation of adenosine in the structures of EF.CaM.cAMP.PP(i) with EF.CaM.3.dATP revealed about 160 degrees rotation in the torsion angle of N-glycosyl bond from the +anti conformation in 3.dATP to -syn in cAMP; such a rotation could serve to distinguish against substrates with the N-2 amino group of purine. The catalytic rate of EF for ITP was about 2 orders of magnitude better than that for GTP, supporting the potential role of this rotation in substrate selectivity of EF. The anomalous difference Fourier map revealed that two ytterbium ions (Yb(3+)) could bind the catalytic site of EF.CaM in the presence of cAMP and PP(i), suggesting the presence of two magnesium ions at the catalytic site of EF. We hypothesize that EF could use a "histidine and two-metal ion" hybrid mechanism to facilitate the cyclization reaction.

Structure of anthrax edema factor-calmodulin-adenosine 5'-(alpha,beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site.

Anthrax edema factor (EF) is a key virulence factor secreted by Bacillus anthracis. Here, we report a structure, at 3.0 A resolution, of the catalytic domain of EF (EF3) in complex with calmodulin (CaM) and adenosine 5'-(alpha,beta-methylene)-triphosphate (AMPCPP). Although the binding of the triphosphate of AMPCPP to EF3 can be superimposed on that of previously determined 3'deoxy-ATP (3'dATP) and 2'deoxy 3' anthraniloyl-ATP (2'd3' ANT-ATP) in EF3-CaM, the ribose and the adenine rings of AMPCPP are rotated approximately 105 and 180 degrees, respectively, relative to those of 3'dATP and 2'd3'ANT-ATP. Based on this model, K382 and F586 should play key roles in the recognition of adenine. However, mutations of these residues to alanine either separately or together cause only modest changes in Michaelis-Menten constants and IC50 values of AMPCPP and cAMP. Therefore, this alternate binding mode of the adenosine of AMPCPP binds to EF likely playing only a minor role in ATP binding and in catalysis.

Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection.

Edema factor (EF), a key virulence factor in anthrax pathogenesis, has calmodulin (CaM)-activated adenylyl cyclase activity. We have found that adefovir dipivoxil, a drug approved to treat chronic infection of hepatitis B virus, effectively inhibits EF-induced cAMP accumulation and changes in cytokine production in mouse primary macrophages. Adefovir diphosphate (PMEApp), the active cellular metabolite of adefovir dipivoxil, inhibits the adenylyl cyclase activity of EF in vitro with high affinity (K(i) = 27 nM). A crystal structure of EF-CaM-PMEApp reveals that the catalytic site of EF forms better van der Waals contacts and more hydrogen bonds with PMEApp than with its endogenous substrate, ATP, providing an explanation for the approximately 10,000-fold higher affinity EF-CaM has for PMEApp versus ATP. Adefovir dipivoxil is a clinically approved drug that can block the action of an anthrax toxin. It can be used to address the role of EF in anthrax pathogenesis.