The herpes simplex virus type 1 DNA polymerase accessory protein, UL42, contains a functional protease-resistant domain

Appleton

Hogle

et al. 2004

J Virol

114

The human cytomegalovirus DNA polymerase contains a catalytic subunit, UL54, and an accessory protein, UL44. Recent studies suggested that UL54 might interact via its extreme C terminus with UL44 (A. Loregian, R. Rigatti, M. Murphy, E. Schievano, G. Palu', and H. S. Marsden, J. Virol. 77:8336-8344, 2003). To address this hypothesis, we quantitatively measured the binding of peptides corresponding to the extreme C terminus of UL54 to UL44 by using isothermal titration calorimetry. A peptide corresponding to the last 22 residues of UL54 was sufficient to bind specifically to UL44 in a 1:1 complex with a dissociation constant of ca. 0.7 M. To define individual residues in this segment that are crucial for interacting with UL44, we engineered a series of mutations in the C-terminal region of UL54. The UL54 mutants were tested for their ability to interact with UL44 by glutathione S-transferase pulldown assays, for basal DNA polymerase activity, and for long-chain DNA synthesis in the presence of UL44. We observed that deletion of the C-terminal segment or substitution of alanine for Leu1227 or Phe1231 in UL54 greatly impaired both the UL54-UL44 interaction in pulldown assays and long-chain DNA synthesis without affecting basal polymerase activity, identifying these residues as important for subunit interaction. Thus, like the herpes simplex virus UL30-UL42 interaction, a few specific side chains in the C terminus of UL54 are crucial for UL54-UL44 interaction. However, the UL54 residues important for interaction with UL44 are hydrophobic and not basic. This information might aid in the rational design of new drugs for the treatment of human cytomegalovirus infection.Most biological processes depend on the coordinated formation of protein-protein interactions. Aside from their importance for viral biology, several interactions between viral proteins have been proposed as attractive targets for antiviral drug discovery, since the exquisite specificity of such cognate interactions affords the possibility of interfering with them in a highly specific and effective manner (for a review, see reference 16). There is a considerable need for new anti-human cytomegalovirus (HCMV) drugs since, although there are agents, most of which target the polymerization activity of the viral DNA polymerase, their use is limited by pharmacokinetic issues, antiviral resistance, and toxicity (4). A potential novel target for anti-HCMV drugs could be the interaction between the two subunits of the viral DNA polymerase.Analogous to the DNA polymerase of other herpesviruses, the HCMV DNA polymerase is composed of a 1,242-residue catalytic subunit, Pol or UL54 (13,14), and the 433-residue accessory protein UL44 (9). The UL54 protein, which is the homolog of herpes simplex virus type 1 (HSV-1) UL30, has DNA-dependent DNA polymerase activity and 3Ј-5Ј exonuclease activity (3,18,20). The UL44 accessory subunit, which is analogous to the HSV-1 UL42 protein, has been shown to bind double-stranded DNA, to specifically interact with UL54, and to stimul...

Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Vol 78 2004 Residues Crucial For Hcmv Ul54-ul44 Interactiomentioning

confidence: 99%

mentioning

confidence: 99%

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Residues of Human Cytomegalovirus DNA Polymerase Catalytic Subunit UL54 That Are Necessary and Sufficient for Interaction with the Accessory Protein UL44

Appleton

Hogle

et al. 2004

J Virol

114

“…With constant amounts of pORF30 and pORF18, the stimulation of pORF30-mediated long-chain DNA synthesis by pORF18 was also time dependent. As reported for long-chain DNA synthesis by the HSV-1 UL30-UL42 complex (17,24), formation of long DNA products by pORF30-pOR18 could be detected after a 5-to 10-min incubation and reached a maximum within 60 min (not shown). Very similar results were obtained when long-chain DNA synthesis by in vitro-expressed pORF30 and pORF18 was assayed in the same manner (data not shown).…”

Section: Analysis Of Ehv-1 Orf30 and Orf18 Translation Productsmentioning

confidence: 53%

Cloning, Expression, and Functional Characterization of the Equine Herpesvirus 1 DNA Polymerase and Its Accessory Subunit

Case

Cancellotti

et al. 2006

J Virol

We report the expression and characterization of the putative catalytic subunit (pORF30) and accessory protein (pORF18) of equine herpesvirus 1 DNA polymerase, which are encoded by open reading frames 30 and 18 and are homologous to herpes simplex virus type 1 UL30 and UL42, respectively. In vitro transcriptiontranslation of open reading frames 30 and 18 generated proteins of 136 and 45 kDa, respectively. In vitroexpressed pORF30 possessed basal DNA polymerase activity that was stimulated by pORF18, as measured by DNA polymerase assays in vitro. Purified baculovirus-expressed pORF30 exhibited DNA polymerase activity similar to that of the in vitro-expressed protein, and baculovirus-expressed pORF18 could stimulate both nucleotide incorporation and long-chain DNA synthesis by pORF30 in a dose-and time-dependent manner. The salt optima for activity of both pORF30 and the holoenzyme were substantially different from those for other herpesvirus DNA polymerases. As demonstrated by yeast two-hybrid assays, pORF30 and pORF18 could physically interact, most likely with a 1:1 stoichiometry. Finally, by mutational analysis of the 1,220-residue pORF30, we demonstrated that the extreme C terminus of pORF30 is important for physical and functional interaction with the accessory protein, as reported for UL30 and other herpesvirus DNA polymerases. In addition, a C-proximal region of pORF30, corresponding to residues 1114 to 1172, is involved in binding to, and stimulation by, pORF18. Taken together, the results indicate that pORF30 and pORF18 are the equine herpesvirus 1 counterparts of herpes simplex virus type 1 UL30 and UL42 and share many, but not all, of their characteristics.Equine herpesvirus 1 (EHV-1) is a pathogen of major importance in horses that can induce a wide spectrum of diseases (1, 45). Like other herpesviruses, EHV-1 establishes a lifelong infection, via a quiescent state known as latency. The outcomes of infection, either following primary infection or reactivation from latency, vary from mild/unapparent respiratory disease to the induction of abortion in pregnant mares and, in extreme cases, neurological disease resulting in paralysis and ultimately death.EHV-1 is classified on biological grounds as a member of the Alphaherpesvirinae, a subfamily of the herpesviruses which is typified by herpes simplex virus type 1 (HSV-1) and also includes varicella-zoster virus (VZV) and pseudorabies virus (PRV) (49). The complete DNA sequence of a pathogenic British isolate (strain Ab4) of EHV-1 was recently determined (56). The genome is approximately 150 kbp in size, is similar in arrangement to the HSV-1, VZV, and PRV genomes, and contains 80 open reading frames (ORFs), corresponding to 76 distinct genes, likely to encode proteins. Comparison of predicted amino acid sequences to those of HSV-1, VZV, and PRV homologs allowed the functions of many EHV-1 proteins to be putatively assigned. However, only a few of these proteins have been studied so far, although the number of characterized proteins is rapidly increasing....

Disruption of protein–protein interactions: Towards new targets for chemotherapy

Journal Cellular Physiology

Palù

2005

101

Protein-protein interactions play a key role in various mechanisms of cellular growth and differentiation, and in the replication of pathogen organisms in host cells. Thus, inhibition of these interactions is a promising novel approach for rational drug design against a wide number of cellular and microbial targets. In the past few years, attempts to inhibit protein-protein interactions using antibodies, peptides, and synthetic or natural small molecules have met with varying degrees of success, and these will be the focus of this review.