scholarly journals Divide et impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 941
Author(s):  
Hanieh Ghassabian ◽  
Federico Falchi ◽  
Martina Timmoneri ◽  
Beatrice Mercorelli ◽  
Arianna Loregian ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Post Infection ◽  
In Silico ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
In Silico Screening ◽  
Processivity Factor

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

scholarly journals Divide et Impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication

2020 ◽  
Author(s):  
Hanieh Ghassabian ◽  
Federico Falchi ◽  
Martina Timmoneri ◽  
Beatrice Mercorelli ◽  
Arianna Loregian ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
In Silico ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
In Silico Screening ◽  
Infected Cells ◽  
Processivity Factor

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets of therapeutic intervention. Among the latter, viral protein-protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication, we performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB40-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited AD169 in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells with B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

scholarly journals The Autoregulatory and Transactivating Functions of the Human Cytomegalovirus IE86 Protein Use Independent Mechanisms for Promoter Binding

2007 ◽  
Vol 81 (11) ◽  
pp. 5807-5818 ◽  
Author(s):  
Dustin T. Petrik ◽  
Kimberly P. Schmitt ◽  
Mark F. Stinski
Keyword(s):  
Human Cytomegalovirus ◽  
Protein Interactions ◽  
Chromatin Immunoprecipitation ◽  
Viral Gene Expression ◽  
Artificial Chromosome ◽  
Viral Gene ◽  
Chip Assay ◽  
Protein Protein Interactions ◽  
Multiple Sequence ◽  
Viral Genes

ABSTRACT The functions of the human cytomegalovirus (HCMV) IE86 protein are paradoxical, as it can both activate and repress viral gene expression through interaction with the promoter region. Although the mechanism for these functions is not clearly defined, it appears that a combination of direct DNA binding and protein-protein interactions is involved. Multiple sequence alignment of several HCMV IE86 homologs reveals that the amino acids 534LPIYE538 are conserved between all primate and nonprimate CMVs. In the context of a bacterial artificial chromosome (BAC), mutation of both P535 and Y537 to alanines (P535A/Y537A) results in a nonviable BAC. The defective HCMV BAC does not undergo DNA replication, although the P535A/Y537A mutant IE86 protein appears to be stably expressed. The P535A/Y537A mutant IE86 protein is able to negatively autoregulate transcription from the major immediate-early (MIE) promoter and was recruited to the MIE promoter in a chromatin immunoprecipitation (ChIP) assay. However, the P535A/Y537A mutant IE86 protein was unable to transactivate early viral genes and was not recruited to the early viral UL4 and UL112 promoters in a ChIP assay. From these data, we conclude that the transactivation and repressive functions of the HCMV IE86 protein can be separated and must occur through independent mechanisms.

Aryloxy Triester Phosphoramidates as Phosphoserine Biocleavable Masking Motifs

2019 ◽  
Author(s):  
Ageo Miccoli ◽  
Binar A. Dhiani ◽  
Peter J. Thornton ◽  
Olivia A. Lambourne ◽  
Edward James ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
In Silico ◽  
Parent Compound ◽  
Cellular Protein ◽  
Carboxypeptidase Y ◽  
Protein Protein Interactions ◽  
Enzymatic Assays ◽  
Specific Targeting

Many cellular protein-protein interactions (PPIs) are mediated by phosphoserine. The specific targeting of these PPIs by phosphoserine-containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinders its uptake into cells. To address these issues, we herein report the masking of the phosphate group of phosphoserine with biocleavable aryloxy triester phosphoramidate groups. A combination of <i>in vitro</i> enzymatic assays and <i>in silico</i> studies, using carboxypeptidase Y and Hint-1 respectively, showed that the phosphate masking groups are metabolized to release phosphoserine. To probe the applicability of this phosphoserine masking approach, it was applied to a phosphoserine-containing inhibitor of 14-3-3 dimerization, and this generated molecules with improved pharmacological activity in cells compared to their unmasked phosphoserine-containing parent compound. Collectively, the data showcases the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups as an efficient intracellular delivery system for phosphoserine-containing molecules.

Aryloxy Triester Phosphoramidates as Phosphoserine Biocleavable Masking Motifs

2019 ◽  
Author(s):  
Ageo Miccoli ◽  
Binar A. Dhiani ◽  
Peter J. Thornton ◽  
Olivia A. Lambourne ◽  
Edward James ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
In Silico ◽  
Parent Compound ◽  
Cellular Protein ◽  
Carboxypeptidase Y ◽  
Protein Protein Interactions ◽  
Enzymatic Assays ◽  
Specific Targeting

Many cellular protein-protein interactions (PPIs) are mediated by phosphoserine. The specific targeting of these PPIs by phosphoserine-containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinders its uptake into cells. To address these issues, we herein report the masking of the phosphate group of phosphoserine with biocleavable aryloxy triester phosphoramidate groups. A combination of <i>in vitro</i> enzymatic assays and <i>in silico</i> studies, using carboxypeptidase Y and Hint-1 respectively, showed that the phosphate masking groups are metabolized to release phosphoserine. To probe the applicability of this phosphoserine masking approach, it was applied to a phosphoserine-containing inhibitor of 14-3-3 dimerization, and this generated molecules with improved pharmacological activity in cells compared to their unmasked phosphoserine-containing parent compound. Collectively, the data showcases the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups as an efficient intracellular delivery system for phosphoserine-containing molecules.

scholarly journals The Alphaviral Capsid Protein Inhibits IRAK1-Dependent TLR Signaling

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 377
Author(s):  
V. Douglas Landers ◽  
Daniel W. Wilkey ◽  
Michael L. Merchant ◽  
Thomas C. Mitchell ◽  
Kevin J. Sokoloski
Keyword(s):  
Capsid Protein ◽  
Protein Interactions ◽  
Viral Gene Expression ◽  
Encephalitis Virus ◽  
Model Systems ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
Equine Encephalitis Virus ◽  
Host Pathogen ◽  
The Impact

Alphaviruses are arthropod-borne RNA viruses which can cause either mild to severe febrile arthritis which may persist for months, or encephalitis which can lead to death or lifelong cognitive impairments. The non-assembly molecular role(s), functions, and protein–protein interactions of the alphavirus capsid proteins have been largely overlooked. Here we detail the use of a BioID2 biotin ligase system to identify the protein–protein interactions of the Sindbis virus capsid protein. These efforts led to the discovery of a series of novel host–pathogen interactions, including the identification of an interaction between the alphaviral capsid protein and the host IRAK1 protein. Importantly, this capsid–IRAK1 interaction is conserved across multiple alphavirus species, including arthritogenic alphaviruses SINV, Ross River virus, and Chikungunya virus; and encephalitic alphaviruses Eastern Equine Encephalitis virus, and Venezuelan Equine Encephalitis virus. The impact of the capsid–IRAK1 interaction was evaluated using a robust set of cellular model systems, leading to the realization that the alphaviral capsid protein specifically inhibits IRAK1-dependent signaling. This inhibition represents a means by which alphaviruses may evade innate immune detection and activation prior to viral gene expression. Altogether, these data identify novel capsid protein–protein interactions, establish the capsid–IRAK1 interaction as a common alphavirus host–pathogen interface, and delineate the molecular consequences of the capsid–IRAK1 interaction on IRAK1-dependent signaling.

scholarly journals Identification of Protein-Protein Interaction Inhibitors Targeting Vaccinia Virus Processivity Factor for Development of Antiviral Agents

2011 ◽  
Vol 55 (11) ◽  
pp. 5054-5062 ◽  
Author(s):  
Norbert Schormann ◽  
Charnell Inglis Sommers ◽  
Mark N. Prichard ◽  
Kathy A. Keith ◽  
James W. Noah ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Effective Interaction ◽  
Antiviral Agents ◽  
Polymerase Activity ◽  
Protein Protein Interactions ◽  
Uracil Dna Glycosylase ◽  
Protein Protein Interaction ◽  
Processivity Factor

ABSTRACTPoxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

scholarly journals The Late Promoter of the Human Cytomegalovirus Viral DNA Polymerase Processivity Factor Has an Impact on Delayed Early and Late Viral Gene Products but Not on Viral DNA Synthesis

2007 ◽  
Vol 81 (12) ◽  
pp. 6197-6206 ◽  
Author(s):  
Hiroki Isomura ◽  
Mark F. Stinski ◽  
Ayumi Kudoh ◽  
Sanae Nakayama ◽  
Satoko Iwahori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Dna ◽  
Recombinant Viruses ◽  
Tata Element ◽  
Processivity Factor

ABSTRACT Transcription of the DNA polymerase processivity factor gene (UL44) of human cytomegalovirus initiates at three distinct start sites, which are differentially regulated during productive infection. Two of these start sites, the distal and proximal sites, are active at early times, and the middle start site is active at only late times after infection (F. Leach and E. S. Mocarski, J. Virol. 63:1783-1791, 1989). Compared to the wild type, UL44 gene expression was lower for recombinant viruses with the distal or the middle TATA element mutated. The transcripts initiating from the distal or middle start site facilitated late viral gene expression. The level of viral DNA synthesis was affected by mutation of the distal TATA element. In contrast, mutation of the middle TATA element did not affect the level of viral DNA synthesis, but it did affect significantly the level of late viral gene expression. Recombinant viruses with the distal or middle TATA element mutated grew more slowly than the wild type at both low and high multiplicities of infection. Reduced expression of the UL44 gene from the late middle viral promoter correlated with decreased late viral protein expression and decreased viral growth.

scholarly journals Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

2020 ◽  
Author(s):  
Hanieh Ghassabian ◽  
Federico Falchi ◽  
Veronica Di Antonio ◽  
Martina Timmoneri ◽  
Beatrice Mercorelli ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Polymerase ◽  
Therapeutic Intervention ◽  
Catalytic Subunit ◽  
Viral Gene ◽  
Drug Resistant ◽  
New Targets ◽  
New Class ◽  
Processivity Factor

ABSTRACTHuman cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the dimerization interface. Subsequently, we performed an in-silico screening to select 18 small molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.IMPORTANCEHCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary infections and reactivation in non-immunocompetent individuals often result in life-threatening conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often limited by toxicity and selection of drug-resistant viral strains, making the identification of new targets of therapeutic intervention crucial for a successful management of HCMV infections. The significance of our work is in identifying the dimerization of the DNA polymerase processivity factor UL44 as an alternative antiviral target. We could show that full length UL44 dimerizes in a cellular context with high affinity and that such interaction could be targeted by small molecules, thus inhibiting the replication of several HCMV strains, including a drug-resistant mutant. Thus, our work could pave the way to the development of a new class of anti-HCMV compounds that act by targeting UL44 dimerization.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals TRIM22. A Multitasking Antiviral Factor

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1864
Author(s):  
Isabel Pagani ◽  
Guido Poli ◽  
Elisa Vicenzi
Keyword(s):  
Protein Interactions ◽  
Influenza A ◽  
Direct Interaction ◽  
Type I Interferons ◽  
Viral Gene ◽  
Target Cells ◽  
Type I ◽  
Protein Protein Interactions ◽  
Viral Genomes ◽  
Dna And Rna

Viral invasion of target cells triggers an immediate intracellular host defense system aimed at preventing further propagation of the virus. Viral genomes or early products of viral replication are sensed by a number of pattern recognition receptors, leading to the synthesis and production of type I interferons (IFNs) that, in turn, activate a cascade of IFN-stimulated genes (ISGs) with antiviral functions. Among these, several members of the tripartite motif (TRIM) family are antiviral executors. This article will focus, in particular, on TRIM22 as an example of a multitarget antiviral member of the TRIM family. The antiviral activities of TRIM22 against different DNA and RNA viruses, particularly human immunodeficiency virus type 1 (HIV-1) and influenza A virus (IAV), will be discussed. TRIM22 restriction of virus replication can involve either direct interaction of TRIM22 E3 ubiquitin ligase activity with viral proteins, or indirect protein–protein interactions resulting in control of viral gene transcription, but also epigenetic effects exerted at the chromatin level.

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