Biomedical subjects

Donald M Coen

Publications and source records attributed to Donald M Coen.

At least 19 recordsLinked to original sources

PubMed2007

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication.

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

Animals↗

PubMed2006

Selective anti-cytomegalovirus compounds discovered by screening for inhibitors of subunit interactions of the viral polymerase.

Better drugs are needed against human cytomegalovirus (HCMV), a pathogen responsible for severe diseases in immunocompromised hosts and newborn children. We investigated whether selective inhibitors of HCMV replication could be discovered by screening for compounds that disrupt the interaction between the accessory subunit of the viral DNA polymerase, UL44, and the C-terminal 22 residues of the catalytic subunit. From approximately 50,000 small molecules, we identified 5 structurally diverse compounds that not only specifically interfere with this interaction, but also with the physical and functional interaction of UL44 with full-length catalytic subunit. These five compounds also inhibited HCMV replication with sub- to low micromolar potency, and at concentrations up to 500-fold lower than those at which they exhibited cytotoxicity. These compounds represent a promising starting point for the development of anti-HCMV drugs.

Antiviral Agents↗

PubMed2006

Low-level expression and reversion both contribute to reactivation of herpes simplex virus drug-resistant mutants with mutations on homopolymeric sequences in thymidine kinase.

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.

Acyclovir↗

PubMed2006

Prediction and identification of herpes simplex virus 1-encoded microRNAs.

MicroRNAs (miRNAs) are key regulators of gene expression in higher eukaryotes. Recently, miRNAs have been identified from viruses with double-stranded DNA genomes. To attempt to identify miRNAs encoded by herpes simplex virus 1 (HSV-1), we applied a computational method to screen the complete genome of HSV-1 for sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. Using this method, we identified 11 HSV-1 genomic loci predicted to encode 13 miRNA precursors and 24 miRNA candidates. Eight of the HSV-1 miRNA candidates were predicted to be conserved in HSV-2. The precursor and the mature form of one HSV-1 miRNA candidate, which is encoded approximately 450 bp upstream of the transcription start site of the latency-associated transcript (LAT), were detected during infection of Vero cells by Northern blot hybridization. These RNAs, which behave as late gene products, are not predicted to be conserved in HSV-2. Additionally, small RNAs, including some that are roughly the expected size of precursor miRNAs, were detected using probes for miRNA candidates derived from sequences encoding the 8.3-kilobase LAT, from sequences complementary to U(L)15 mRNA, and from the region between ICP4 and U(S)1. However, no species the size of typical mature miRNAs were detected using these probes. Three of these latter miRNA candidates were predicted to be conserved in HSV-2. Thus, HSV-1 encodes at least one miRNA. We hypothesize that HSV-1 miRNAs regulate viral and host gene expression.

Animals↗

PubMed2006

Virucidal activity of a GT-rich oligonucleotide against herpes simplex virus mediated by glycoprotein B.

We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4 degrees C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of pre-attached virions into cells at 37 degrees C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 microM ISIS 5652 at 37 degrees C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4 degrees C or with an unrelated oligonucleotide at 37 degrees C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.

Animals↗

PubMed2005

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.

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 Ile(135) 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.

Catalysis↗

PubMed2005

Herpesviral latency-associated transcript gene promotes assembly of heterochromatin on viral lytic-gene promoters in latent infection.

Herpes simplex virus (HSV) persists in its human host and evades the immune response by undergoing a latent infection in sensory neurons, from which it can reactivate periodically. HSV expresses >80 gene products during productive ("lytic") infection, but only the latency-associated transcript (LAT) gene is expressed at abundant levels during latent infection. The LAT gene has been shown to repress lytic-gene expression in sensory neurons. In this study, we use chromatin immunoprecipitation to show that HSV lytic-gene promoters become complexed with modified histones associated with heterochromatin during the course of establishment of latent infection. Experiments comparing LAT-negative and LAT-positive viruses show that a function encoded by the LAT gene increases the amount of dimethyl lysine 9 form of histone H3 or heterochromatin and reduces the amount of dimethyl lysine 4 form of histone H3, a part of active chromatin, on viral lytic-gene promoters. Thus, HSV, and in particular the HSV LAT gene, may manipulate the cellular histone modification machinery to repress its lytic-gene expression and contribute to the persistence of its genome in a quiescent form in sensory neurons.

Animals↗

PubMed2005

Binding of herpes simplex virus-1 US11 to specific RNA sequences.

Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.

Base Sequence↗

PubMed2005

An unusual internal ribosome entry site in the herpes simplex virus thymidine kinase gene.

We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion in the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5' cap or had a stable stem loop 5' of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-eIF2-GTP/Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the single-base deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs.

Animals↗

PubMed2005

A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress.

Most protein phosphatases have little intrinsic substrate specificity, making selective pharmacological inhibition of specific dephosphorylation reactions a challenging problem. In a screen for small molecules that protect cells from endoplasmic reticulum (ER) stress, we identified salubrinal, a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Salubrinal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. These results suggest that selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, salubrinal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.

Animals↗

PubMed2005

Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis.

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.

Amino Acid Sequence↗

PubMed2005

Herpes simplex virus 1 immediate-early and early gene expression during reactivation from latency under conditions that prevent infectious virus production.

The program of gene expression exhibited by herpes simplex virus during productive infection of cultured cells is well established; however, less is known about the regulatory controls governing reactivation from latency in neurons. One difficulty in examining gene regulation during reactivation lies in distinguishing between events occurring in initial reactivating cells versus events occurring in secondarily infected cells. Thus, two inhibitors were employed to block production of infectious virus: acyclovir, which inhibits viral DNA synthesis, and WAY-150138, which permits viral DNA synthesis but inhibits viral DNA encapsidation. Latently infected murine ganglia were explanted in the presence of either inhibitor, and then amounts of RNA, DNA, or infectious virus were quantified. In ganglia explanted for 48 h, the levels of five immediate-early and early RNAs did not exhibit meaningful differences between acyclovir and WAY-150138 treatments when analyzed by in situ hybridization or quantitative reverse transcription-PCR. However, comparative increases in viral DNA and RNA content in untreated ganglia suggested that virus was produced before 48 h postexplant. This was confirmed by the detection of infectious virus as early as 14 h postexplant. Together, these results suggest that high levels of viral gene expression at 48 h postexplant are due largely to the production of infectious virus and subsequent spread through the tissue. These results lead to a reinterpretation of previous results indicating a role for DNA replication in immediate-early and early viral gene expression; however, it remains possible that viral gene expression is regulated differently in neurons than in cultured cells.

Acyclovir↗

PubMed2004

Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of herpes simplex virus 1 infection.

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1alpha, MIP-1beta and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.

Animals↗

PubMed2004

The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer.

The human cytomegalovirus DNA polymerase consists of a catalytic subunit, UL54, and a presumed processivity factor, UL44. We have solved the crystal structure of residues 1-290 of UL44 to 1.85 A resolution by multiwavelength anomalous dispersion. The structure reveals a dimer of UL44 in the shape of a C clamp. Each monomer of UL44 shares its overall fold with other processivity factors, including herpes simplex virus UL42, which is a monomer that binds DNA directly, and the sliding clamp, PCNA, which is a trimer that surrounds DNA, although these proteins share no obvious sequence homology. Analytical ultracentrifugation and gel filtration measurements demonstrated that UL44 also forms a dimer in solution, and substitution of large hydrophobic residues along the homodimer interface with alanine disrupted dimerization and decreased DNA binding. UL44 represents a hybrid processivity factor as it binds DNA directly like UL42, but forms a C clamp that may surround DNA like PCNA.

Binding Sites↗

PubMed2004

Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) ordinarily exists in electrophoretically distinct hypophosphorylated and hyperphosphorylated forms. Human cytomegalovirus infection induced forms of this subunit whose electrophoretic mobilities were intermediate without decreases in abundance of the original forms. Phosphatase treatment nearly eliminated the intermediate migrating forms. In vitro, the viral protein kinase, UL97, phosphorylated this subunit, a recombinant protein containing the CTD, and peptides containing the CTD consensus sequence, YSPTSPS. Phosphorylation occurred predominantly on serine 5 and was substantially reduced when either serine 2 or 5 was already phosphorylated. The abundance of the intermediate and hypophosphorylated forms was reduced at most twofold during infections in which UL97 was genetically or pharmacologically inhibited. These results identify a new pattern of RNA polymerase II modification induced by virus infection and a viral enzyme that phosphorylates the CTD in vitro, but only possibly in vivo.

Cell Line↗

PubMed2004

The herpes simplex virus processivity factor, UL42, binds DNA as a monomer.

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer in solution. However, UL42 is structurally similar to sliding clamp processivity factors, such as PCNA, which encircle DNA as a multimeric ring. We used chemical crosslinking and electrophoretic mobility-shift assays to investigate whether UL42 oligomerizes upon DNA binding. UL42 did not form intermolecular crosslinks upon treatment with glutaraldehyde in the presence of DNA, whereas proteins that are known to be multimers in solution were successfully crosslinked by this treatment. This result suggests that UL42 does not form multimers on DNA. We next analyzed the composition of UL42:DNA complexes using electrophoretic mobility-shift assays. UL42 was mixed with a maltose-binding protein-UL42 fusion protein before being added to DNA. The patterns of electrophoretic mobility of the resultant protein:DNA complexes were those predicted if each isoform of UL42 binds to DNA as a monomer. From this result and the failure of UL42 to form crosslinks, we infer that UL42 binds DNA as a monomer.

Carrier Proteins↗

PubMed2004

Identification of a small molecule that inhibits herpes simplex virus DNA Polymerase subunit interactions and viral replication.

The interaction between the catalytic subunit Pol and the processivity subunit UL42 of herpes simplex virus DNA polymerase has been characterized structurally and mutationally and is a potential target for novel antiviral drugs. We developed and validated an assay for small molecules that could disrupt the interaction of UL42 and a Pol-derived peptide and used it to screen approximately 16,000 compounds. Of 37 "hits" identified, four inhibited UL42-stimulated long-chain DNA synthesis by Pol in vitro, of which two exhibited little inhibition of polymerase activity by Pol alone. One of these specifically inhibited the physical interaction of Pol and UL42 and also inhibited viral replication at concentrations below those that caused cytotoxic effects. Thus, a small molecule can inhibit this protein-protein interaction, which provides a starting point for the discovery of new antiviral drugs.

Animals↗

PubMed2004

Residues of human cytomegalovirus DNA polymerase catalytic subunit UL54 that are necessary and sufficient for interaction with the accessory protein UL44.

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 microM. 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.

Amino Acid Sequence↗