scholarly journals Inhibition of Human Cytomegalovirus DNA Polymerase by C-Terminal Peptides from the UL54 Subunit

2003 ◽  
Vol 77 (15) ◽  
pp. 8336-8344 ◽  
Author(s):  
Arianna Loregian ◽  
Roberto Rigatti ◽  
Mary Murphy ◽  
Elisabetta Schievano ◽  
Giorgio Palu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Helical Structure ◽  
Structural Similarity ◽  
Accessory Protein ◽  
Physical Interaction ◽  
Virus Growth ◽  
C Terminus ◽  
Simplex Virus ◽  
Carboxy Terminal

ABSTRACT In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially α-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.

scholarly journals Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells

2008 ◽  
Vol 82 (24) ◽  
pp. 12574-12579 ◽  
Author(s):  
Elisa Sinigalia ◽  
Gualtiero Alvisi ◽  
Beatrice Mercorelli ◽  
Donald M. Coen ◽  
Gregory S. Pari ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Accessory Protein ◽  
C Terminus ◽  
Cellular Context ◽  
In Cells

ABSTRACT The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy.

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

2004 ◽  
Vol 78 (1) ◽  
pp. 158-167 ◽  
Author(s):  
Arianna Loregian ◽  
Brent A. Appleton ◽  
James M. Hogle ◽  
Donald M. Coen
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Rational Design ◽  
Polymerase Activity ◽  
Catalytic Subunit ◽  
New Drugs ◽  
Accessory Protein ◽  
C Terminus ◽  
Long Chain

ABSTRACT 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 μM. 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.

scholarly journals Specific Residues in the Connector Loop of the Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 Are Crucial for Interaction with the UL54 Catalytic Subunit

2004 ◽  
Vol 78 (17) ◽  
pp. 9084-9092 ◽  
Author(s):  
Arianna Loregian ◽  
Brent A. Appleton ◽  
James M. Hogle ◽  
Donald M. Coen
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Catalytic Subunit ◽  
Accessory Protein ◽  
Simplex Virus ◽  
Long Chain

ABSTRACT The human cytomegalovirus DNA polymerase includes an accessory protein, UL44, which has been proposed to act as a processivity factor for the catalytic subunit, UL54. How UL44 interacts with UL54 has not yet been elucidated. The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30. To investigate the role of the UL44 connector loop, we replaced each of its amino acids (amino acids 129 to 140) with alanine. We then tested the effect of each substitution on the UL44-UL54 interaction by glutathione S-transferase pulldown and isothermal titration calorimetry assays, on the stimulation of UL54-mediated long-chain DNA synthesis by UL44, and on the binding of UL44 to DNA-cellulose columns. Substitutions that affected residues 133 to 136 of the connector loop measurably impaired the UL44-UL54 interaction without altering the ability of UL44 to bind DNA. One substitution, I135A, completely disrupted the binding of UL44 to UL54 and inhibited the ability of UL44 to stimulate long-chain DNA synthesis by UL54. Thus, similar to the herpes simplex virus UL30-UL42 interaction, a residue of the connector loop of the accessory subunit is crucial for UL54-UL44 interaction. However, while alteration of a polar residue of the UL42 connector loop only partially reduced binding to UL30, substitution of a hydrophobic residue of UL44 completely disrupted the UL54-UL44 interaction. This information may aid the discovery of small-molecule inhibitors of the UL44-UL54 interaction.

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

1993 ◽  
Vol 74 (10) ◽  
pp. 2181-2189 ◽  
Author(s):  
R. K. Hamatake ◽  
M. Bifano ◽  
D. J. Tenney ◽  
W. W. Hurlburt ◽  
M. G. Cordingley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Accessory Protein ◽  
Simplex Virus

scholarly journals Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase.

1997 ◽  
Vol 41 (12) ◽  
pp. 2680-2685 ◽  
Author(s):  
D J Tenney ◽  
G Yamanaka ◽  
S M Voss ◽  
C W Cianci ◽  
A V Tuomari ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Potent Inhibitor ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv Thymidine Kinase

Lobucavir (LBV) is a deoxyguanine nucleoside analog with broad-spectrum antiviral activity. LBV was previously shown to inhibit herpes simplex virus (HSV) DNA polymerase after phosphorylation by the HSV thymidine kinase. Here we determined the mechanism of action of LBV against human cytomegalovirus (HCMV). LBV inhibited HCMV DNA synthesis to a degree comparable to that of ganciclovir (GCV), a drug known to target the viral DNA polymerase. The expression of late proteins and RNA, dependent on viral DNA synthesis, was also inhibited by LBV. Immediate-early and early HCMV gene expression was unaffected, suggesting that LBV acts temporally coincident with HCMV DNA synthesis and not through cytotoxicity. In vitro, the triphosphate of LBV was a potent inhibitor of HCMV DNA polymerase with a Ki of 5 nM. LBV was phosphorylated to its triphosphate form intracellularly in both infected and uninfected cells, with phosphorylated metabolite levels two- to threefold higher in infected cells. GCV-resistant HCMV isolates, with deficient GCV phosphorylation due to mutations in the UL97 protein kinase, remained sensitive to LBV. Overall, these results suggest that LBV-triphosphate halts HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. Furthermore, LBV may be effective in the treatment of GCV-resistant HCMV.

scholarly journals A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1

2016 ◽  
Vol 90 (23) ◽  
pp. 10875-10885 ◽  
Author(s):  
Yi Zheng ◽  
Subodh Kumar Samrat ◽  
Haidong Gu
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Cell Protein ◽  
Physical Interaction ◽  
Herpes Simplex Virus 1 ◽  
C Terminus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTInfected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an α gene product required for viral replication at low multiplicities of infection. Upon entry, nuclear domain 10 (ND10) converges at the incoming DNA and represses viral gene expression. ICP0 contains a RING-type E3 ubiquitin ligase that degrades the ND10 organizer PML and disperses ND10 to alleviate the repression. In the present study, we focused on understanding the regulation of ICP0 E3 ligase activity in the degradation of different ICP0 substrates. We report the following. (i) A SUMO interaction motif located at ICP0 residues 362 to 364 is required for the degradation of PML isoforms II, IV, and VI but not isoform I. This differentiation mechanism exists in both HEp-2 and U2OS cells, regardless of the cell's permissiveness to the ICP0-null virus. (ii) Physical interaction between SIM362–364and PML II is necessary but not sufficient for PML II degradation. Both proximal sequences surrounding SIM362–364and distal sequences located at the ICP0 C terminus enhance the degradation of PML II. (iii) The ICP0 C terminus is dispensable for PML I degradation. Instead, bipartite PML I binding domains located in the N-terminal half of ICP0 coordinate to promote the degradation of PML I. (iv) The stability of ICP0, but not its ND10 fusion ability, affects the rate of PML I degradation. Taken together, our results show that ICP0 uses at least two regulatory mechanisms to differentiate its substrates. The disparate recognition of the ICP0 E3 substrates may be related to the different roles these substrates may play in HSV-1 infection.IMPORTANCEViruses have a limited genetic coding capacity but must encounter a multilayered comprehensive host defense. To establish a successful infection, viruses usually produce multifunctional proteins to coordinate the counteractions. Here we report that an HSV-1 protein, ICP0, can recognize individual host factors and target them differently for destruction. We identified elements that are important for the ICP0 E3 ubiquitin ligase to differentially recognize two of its substrates, PML I and PML II. This is the first study that has systematically investigated how ICP0 discriminates two similar molecules by very different mechanisms. This work lays the foundation for understanding the role of host defensive factors and the mechanisms viruses use to take advantage of some host proteins while destroying others.

scholarly journals Identification of Crucial Hydrogen-Bonding Residues for the Interaction of Herpes Simplex Virus DNA Polymerase Subunits via Peptide Display, Mutational, and Calorimetric Approaches

2001 ◽  
Vol 75 (11) ◽  
pp. 4990-4998 ◽  
Author(s):  
Kristie Grove Bridges ◽  
Connie S. Chow ◽  
Donald M. Coen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Antiviral Drug ◽  
Titration Calorimetry ◽  
Random Peptide ◽  
C Terminus ◽  
Simplex Virus ◽  
Mutant Forms ◽  
Peptide Display

ABSTRACT The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 μM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.

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