Expression of the Catalytic Subunit (UL54) and the Accessory Protein (UL44) of Human Cytomegalovirus DNA Polymerase in a Coupledin VitroTranscription/Translation System

doi:10.1006/prep.1997.0781

Protein Expression and Purification

Volume 11, Issue 2, November 1997, Pages 209-218

https://doi.org/10.1006/prep.1997.0781 Get rights and content

Abstract

The catalytic subunit (UL54) and accessory protein (UL44) of human cytomegalovirus (HCMV) DNA polymerase have been cloned and expressed in anin vitro-coupled transcription/translation reticulocyte lysate system. The influence of the 5′-untranslated region (5′-UTR) on the efficiency of expression from the circular plasmids has been investigated. For expression of both UL54 and UL44, a truncated form of the alfalfa mosaic virus (AMV) RNA4 5′-UTR was found to be superior to the full-length AMV 5′-UTR or the original HCMV 5′-UTRs of different lengths. Protein products withM_r≈ 140 and 55 kDa were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis in the UL54 and UL44in vitroexpression reactions, respectively. The properties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3′–5′ exonuclease activities of the expressed UL54/UL44 complex were found to be dependent on salt concentration in the same manner as the activities of the native enzyme. Thein vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidofovir diphosphate, ganciclovir triphosphate, and foscarnet). This straightforward method for protein expression may also be applicable to other enzymes where reproducible generation of fully functional products is desirable.

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This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC₅₀ values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC₅₀ values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC₅₀ increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC₅₀ increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC₅₀ increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.
Guanine α-carboxy nucleoside phosphonate (G-α-CNP) shows a different inhibitory kinetic profile against the DNA polymerases of human immunodeficiency virus (HIV) and herpes viruses
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Citation Excerpt :
For the experiments in which the Ki values of the test compounds were determined with respect to the template/primers, appropriate concentrations of the template/primers were used in the presence of a fixed concentration of 2.5 µM [3H]dGTP. The pGEM3Z-CMV UL54 plasmid for expression of the catalytic subunit (UL54 protein) of HCMV DNA polymerase was a generous gift from T. Cihlar (Gilead Sciences, Foster City, CA) [35]. Protein expression was performed with the TnT® SP6 Quick Coupled Transcription/Translation System (Promega) [36].
α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class of nucleotide-competing reverse transcriptase (RT) inhibitors (NcRTIs). They were designed to act directly against HIV-1 RT without the need for prior activation (phosphorylation). In this respect, they differ from the nucleoside or nucleotide RTIs [N(t)RTIs] that require conversion to their triphosphate forms before being inhibitory to HIV-1 RT. The guanine derivative (G-α-CNP) has now been synthesized and investigated for the first time. The (L)-(+)-enantiomer of G-α-CNP directly and competitively inhibits HIV-1 RT by interacting with the substrate active site of the enzyme. The (D)-(−)-enantiomer proved inactive against HIV-1 RT. In contrast, the (+)- and (−)-enantiomers of G-α-CNP inhibited herpes (i.e. HSV-1, HCMV) DNA polymerases in a non- or uncompetitive manner, strongly indicating interaction of the (L)-(+)- and the (D)-(−)-G-α-CNPs at a location different from the polymerase substrate active site of the herpes enzymes. Such entirely different inhibition profile of viral polymerases is unprecedented for a single antiviral drug molecule. Moreover, within the class of α-CNPs, subtle differences in their sensitivity to mutant HIV-1 RT enzymes were observed depending on the nature of the nucleobase in the α-CNP molecules. The unique properties of the α-CNPs make this class of compounds, including G-α-CNP, direct acting inhibitors of multiple viral DNA polymerases.
Development and validation of a non-radioactive DNA polymerase assay for studying cytomegalovirus resistance to foscarnet
2007, Journal of Virological Methods
Phenotypic characterisation of the human cytomegalovirus (HCMV) pUL54 DNA polymerase is a useful tool for testing for mutations in the UL54 gene thought to render HCMV resistant to foscarnet. In this study, an in-house non-isotopic method for assessing polymerase enzymatic activity in the presence and absence of foscarnet was developed and its utility for HCMV polymerase phenotyping evaluated. Polymerase activity was assessed by monitoring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain and foscarnet concentrations inhibiting enzymatic activity by 50% were determined. HCMV DNA polymerases were synthesised in vitro by expression of UL54 under the control of the T7 promoter. Mutations of interest were introduced into the wild-type UL54 gene by site-directed mutagenesis. Mutated polymerases and polymerases from HCMV reference strains were studied. The activity of polymerases containing mutations known to confer resistance to foscarnet (V715M, T700A and N495K) was inhibited by concentrations of foscarnet eight to 14 times higher than those required to inhibit wild-type polymerases. Our in-house non-radioactive phenotypic assay was sensitive and reproducible. It is also easy to perform and could provide a convenient method for characterising mutations conferring resistance to foscarnet in HCMV.
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Based on our previous experience with arylsulfone derivatives displaying antiherpetic activity, we synthesized several analogues in which the sulfonyl group is part of a bicyclic structure. The benzene-fused derivative 2H-3-(4-chlorophenyl)-3,4-dihydro-1,4-benzo-thiazine-2-carbonitrile 1,1-dioxide and its thiophene-fused analogue were shown to have favorable activity and selectivity against the betaherpesviruses human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) and 7 (HHV-7). The benzene-fused derivative retained its anti-HCMV activity when evaluated against virus strains resistant to foscarnet, ganciclovir, and/or cidofovir. The compound conferred ≥95% inhibition of viral DNA synthesis in HHV-6-infected cells. RT-PCR analysis of immediate-early, early and late gene products revealed that this arylsulfone compound acts at a step preceding late gene expression, and coinciding with the inhibition exerted by foscarnet. No inhibitory effect was seen in an enzyme assay for DNA elongation catalyzed by the HCMV or HHV-6 DNA polymerase catalytic subunit. The arylsulfone derivatives had no effect on the functional interaction between the catalytic subunit of HCMV DNA polymerase and its accessory protein, nor did they disrupt the physical interaction between the two proteins. We conclude that these arylsulfone derivatives represent new betaherpesvirus inhibitors with a novel mode of action that results in indirect inhibition of viral DNA synthesis.
Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit: Differences in structure and function relative to unliganded UL44
2006, Journal of Biological Chemistry
The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the “connector loop” of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic “plug” from the UL54 peptide and Ile¹³⁵ of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-to-head dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.
A novel colorimetric test to study the susceptibility of human cytomegalovirus DNA polymerase to foscarnet
2005, Pathologie Biologie
Nous décrivons une nouvelle méthode colorimétrique pour déterminer le phénotype de l'ADN polymérase du cytomégalovirus humain (CMV) en mesurant l'incorporation de nucléotides marqués à la digoxigénine dans le brin d'ADN en cours de synthèse. La mutation N495K et l'association des mutations K415R et S291P, observées sur des isolats cliniques résistants au foscarnet ont été étudiées. Les mutations V715M et E756K, connues pour conférer une résistance au foscarnet, ont été choisies comme témoins. Toutes les mutations ont été introduites par mutagenèse dirigée dans le gène UL54 de la souche de référence Towne, préalablement cloné dans un vecteur d'expression. Les polymérases mutées et sauvages ont été synthétisées dans un lysat de réticulocytes à l'aide d'une trousse de transcription–traduction couplée in vitro. L'activité polymérase a été mesurée en présence et en absence de foscarnet. Les concentrations de foscarnet inhibant de 50 % l'activité des polymérases portant la mutation V715M ou E756K sont respectivement 70 et 30 fois supérieures à celle inhibant l'activité de la souche de référence sensible. Les modifications N495K et l'association S291P et K415R induisent une diminution de la sensibilité de la polymérase au foscarnet. Les résultats obtenus avec le test colorimétrique concordent avec ceux obtenus par la technique en radioactivité habituellement utilisée. Cette nouvelle méthode phénotypique pourrait être utile pour caractériser les mutations du gène de l'ADN polymérase du CMV susceptibles de conférer une résistance au foscarnet.
We described a colorimetric method to determine the biochemical phenotype of wild-type and mutated cytomegalovirus (HCMV) DNA polymerases by measuring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. Mutations V715M and E756K, which are known to confer foscarnet-resistance, were used as controls. Mutation N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, were studied. The mutations were introduced by site-directed mutagenesis into wild-type gene UL54 cloned in an expression vector and then polymerases were synthesised by using a commercially available coupled transcription–translation system. The polymerase activity was measured with and without foscarnet. The activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and combination of K415R and S291P, induced a five- and ten-fold decrease in susceptibility to foscarnet, respectively. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. Therefore, this novel phenotypic method could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.

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Regular ArticleExpression of the Catalytic Subunit (UL54) and the Accessory Protein (UL44) of Human Cytomegalovirus DNA Polymerase in a Coupledin VitroTranscription/Translation System☆

Abstract

Int. J. Antimicrob. Agents

Biochem. Pharmacol.

Pharmac. Ther.

Virology

Cell

Virus Res.

Biochim. Biophys. Acta

Biochem. Pharmacol.

Cell

Cell

J. Biol. Chem.

FEBS Letts.

Cytomegalovirus

Fields Virology

Cytomegalovirus: Biology and Infection

Cidofovir, a new agent with potent anti-herpesvirus activity

Antiviral Chemistry Chemother.

Regular Article
Expression of the Catalytic Subunit (UL54) and the Accessory Protein (UL44) of Human Cytomegalovirus DNA Polymerase in a Coupledin VitroTranscription/Translation System☆