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D M Coen

Publications and source records attributed to D M Coen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Accumulation of viral transcripts and DNA during establishment of latency by herpes simplex virus.

Latent infection of mice with wild-type herpes simplex virus is established during an acute phase of ganglionic infection in which there is abundant viral replication and productive-cycle gene expression. Thymidine kinase-negative mutants establish latent infections but are severely impaired for acute ganglionic replication and productive-cycle gene expression. Indeed, by in situ hybridization assays, acute infection by these mutants resembles latency. To assess events during establishment of latency by wild-type and thymidine kinase-negative viruses, we quantified specific viral nucleic acid sequences in mouse trigeminal ganglia during acute ganglionic infection by using sensitive PCR-based assays. Through 32 h postinfection, viral DNA and transcripts representative of the three kinetic classes of productive-cycle genes accumulated to comparable levels in wild-type- and mutant-infected ganglia. At 48 and 72 h, although latency-associated transcripts accumulated to comparable levels in ganglia infected with wild-type or mutant virus, levels of DNA accumulating in wild-type-infected ganglia exceeded those in mutant-infected ganglia by 2 to 3 orders of magnitude. Coincident with this increase in DNA, wild-type-infected ganglia exhibited abundant expression of productive-cycle genes and high titers of infectious progeny. Nevertheless, the levels of productive-cycle RNAs expressed by mutant virus during acute infection greatly exceeded those expressed by wild-type virus during latency. The results thus distinguish acute infection of ganglia by a replication-compromised mutant from latent infection and may have implications for mechanisms of latency.

Animals↗
PubMed1998

Human thymidine kinase can functionally replace herpes simplex virus type 1 thymidine kinase for viral replication in mouse sensory ganglia and reactivation from latency upon explant.

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to approximately 5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.

Animals↗
PubMed1997

The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines.

The product of the human cytomegalovirus (CMV) UL97 gene, which controls ganciclovir phosphorylation in virus-infected cells, is homologous to known protein kinases but diverges from them at a number of positions that are functionally important. To investigate UL97, we raised an antibody against it and overexpressed it in baculovirus-infected insect cells. Recombinant baculovirus expressing full-length UL97 directed the phosphorylation of ganciclovir in insect cells, which was abolished by a four-codon deletion that confers ganciclovir resistance to CMV. When incubated with [gamma-32P]ATP, full-length UL97 was phosphorylated on serine and threonine residues. Phosphorylation was severely impaired by a point mutation that alters lysine-355 in a motif that aligns with subdomain II of protein kinases. However, phosphorylation was impaired much less severely by the four-codon deletion. A UL97 fusion protein expressed from recombinant baculovirus was purified to near homogeneity. It too was phosphorylated upon incubation with [gamma-32P]ATP in vitro. This phosphorylation, which was abolished by the lysine 355 mutation, was optimal at high NaCl and high pH. The activity required either Mn2+ or Mg2+, with a preference for Mn2+, and utilized either ATP or GTP as a phosphate donor, with Kms of 2 and 4 microM, respectively. The phosphorylation rate was first order with protein concentration, consistent with autophosphorylation. These data strongly argue that UL97 is a serine/threonine protein kinase that autophosphorylates and suggest that the four-codon deletion affects its substrate specificity.

Animals↗
PubMed1997

Effects of mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene on enzyme activities, viral replication, and replication fidelity.

The herpes simplex virus DNA polymerase catalytic subunit, which has intrinsic polymerase and 3'-5' exonuclease activities, contains sequence motifs that are homologous to those important for 3'-5' exonuclease activity in other polymerases. The role of one such motif, Exo III, was examined in this study. Mutated polymerases containing either a single tyrosine-to-histidine change at residue 577 or this change plus an aspartic acid-to-alanine at residue 581 in the Exo III motif exhibited defective or undetectable exonuclease activity, respectively, yet retained substantial polymerase activity. Despite the defects in exonuclease activity, the mutant polymerases were able to support viral replication in transient complementation assays, albeit inefficiently. Viruses replicated via the action of these mutant polymerases exhibited substantially increased frequencies of mutants resistant to ganciclovir. Furthermore, when the Exo III mutations were incorporated into the viral genome, the resulting mutant viruses displayed only modestly defect in replication in Vero cells and exhibited substantially increased mutation frequencies. The results suggest that herpes simplex virus can replicate despite severely impaired exonuclease activity and that the 3'-5' exonuclease contributes substantially to the fidelity of viral DNA replication.

Amino Acid Sequence↗
PubMed1997

A viral function represses accumulation of transcripts from productive-cycle genes in mouse ganglia latently infected with herpes simplex virus.

Latent infections of neurons by herpes simplex virus form reservoirs of recurrent viral infections that resist cure. In latently infected neurons, viral gene expression is severely repressed; only the latency-associated transcripts (LATs) are expressed abundantly. Using sensitive reverse transcriptase PCR assays, we analyzed the effects of a deletion mutation in the LAT locus on viral gene expression in latently infected mouse trigeminal ganglia. The deletion mutation, which reduced expression of the major LATs 10(5)-fold, resulted in a approximately 5-fold increase in accumulation of transcripts from the immediate-early gene encoding ICP4, an essential transactivator of viral gene expression. The LAT deletion also resulted in a >10-fold increase in the accumulation of transcripts from the early gene encoding thymidine kinase, whose expression during productive infection stringently depends on ICP4, and positively affected the correlation of the levels of these transcripts with the levels of ICP4 transcripts. We also detected transcripts antisense to ICP4 RNA, which were in substantial excess to ICP4 transcripts in ganglia latently infected with wild-type virus. In contrast to its effects on productive-cycle transcripts, the LAT deletion reduced the accumulation of these antisense transcripts approximately 15-fold. Thus, a viral function associated with the LAT locus represses the accumulation of transcripts from at least two productive-cycle genes in latently infected mouse ganglia. We discuss possible mechanisms and consequences of this repression.

Animals↗
PubMed1996

Translational recoding induced by G-rich mRNA sequences that form unusual structures.

We investigated a herpesvirus mutant that contains a single base insertion in its thymidine kinase (tk) gene yet expresses low levels of TK via a net +1 translational recoding event. Within this mutant gene, we defined a G-rich signal that is sufficient to induce recoding. Unlike other translational recoding events, downstream RNA structures or termination codons did not stimulate recoding, and paused ribosomes were not detected. Mutational analysis indicated that specific tRNAs or codon-anticodon slippage were unlikely to account for recoding. Rather, recoding efficiency correlated with the G-richness of the signal and its ability to form unusual structures. These findings identify a mechanism of translational recoding with unique features and potential implications for clinical drug resistance and other biological systems.

Amino Acid Sequence↗
PubMed1996

Regulation of neighboring gene expression by the herpes simplex virus type 1 thymidine kinase gene.

The herpes simplex virus type 1 thymidine kinase (tk) gene (UL23) lies upstream of the gH (UL22) gene with its 3' end overlapping the gH promoter, and it overlaps the UL24 gene's regulatory and coding sequences at its 5' end in a head-to-head orientation. Thus, tk expression could affect gH expression by promoter occlusion and UL24 expression by transcriptional or posttranscriptional mechanisms. To investigate these possibilities, we analyzed the effects of tk promoter mutations that reduce tk expression on gH and UL24 expression. For gH, tk promoter mutations did not increase the accumulation of gH mRNA or the rate of gH transcription. Thus, tk expression does not appear to down-regulate gH expression. In contrast, we found that decreased tk expression correlated with increased accumulation of UL24 mRNA, particularly a 1.4-kb transcript, at early times postinfection during peak expression of tk, but not at late times when tk mRNA levels have fallen. Results from viral co-infection experiments indicated that down-regulation of UL24 mRNA accumulation requires tk expression in cis. Nuclear run-off experiments did not detect differences in UL24 transcription rates in the mutant viruses. Although we cannot completely exclude a transcriptional mechanism for this down-regulation, these results can be explained by an antisense RNA mechanism acting preferentially in cis.

Animals↗
PubMed1996

Temporal regulation of herpes simplex virus type 1 UL24 mRNA expression via differential polyadenylation.

Using Northern blot, primer extension, and S1 nuclease analyses of wild-type and deletion-containing herpes simplex type 1 viruses, we found that UL24 sequences are contained in six different transcripts that originate from three previously identified mRNA start sites. Thus, the six UL24 transcripts represent three different pairs of 5' coterminal mRNAs. Each transcript pair consists of a short species whose 3' end corresponds to a polyadenylation signal located just downstream of the UL24 open reading frame, and a longer species whose 3' end corresponds to a polyadenylation signal located downstream of the UL26 gene. Maximal accumulation of the short UL24 transcripts was at early times during infection, while accumulation of the longer species did not decrease at late times. Consistent with early kinetics, the short transcripts were less sensitive to drugs that inhibited viral DNA replication than the longer transcripts which exhibited leaky-late kinetics. Quantitative S1 nuclease analysis indicated that 3' ends corresponding to the UL24 polyadenylation site were significantly more abundant at early times than at late times. Thus, differential polyadenylation determines the kinetics of accumulation of different UL24 transcripts.

Animals↗
PubMed1995

Specific inhibition of herpes simplex virus DNA polymerase by helical peptides corresponding to the subunit interface.

The herpes simplex virus DNA polymerase consists of two subunits--a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial alpha-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of alpha-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase delta and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

Amino Acid Sequence↗
PubMed1995

Synergistic effects on ganglionic herpes simplex virus infections by mutations or drugs that inhibit the viral polymerase and thymidine kinase.

Herpes simplex virus encodes proteins, such as DNA polymerase, that are essential for its replication and proteins, such as thymidine kinase, that are not essential for replication in cell culture, but are important for pathogenesis in animal models. However, certain mutations affecting these proteins exert little or no effect on replication or pathogenesis. We tested the effects of combining two such mutations--one that alters DNA polymerase and one that decreases but does not abolish thymidine kinase activity--on replication in cultured cells and on acute and latent infections in mice. The double mutant replicated similarly to the single mutants and wild-type virus both in cell culture and acutely in the mouse eye. However, it was severely impaired for acute replication in trigeminal ganglia and for reactivatable latent infections. This impairment depended upon the polymerase mutation. Similarly, although Ro 31-5140, a thymidine kinase inhibitor, did not potentiate the antiviral effects of phosphonoacetic acid, a polymerase inhibitor, in cell culture, the two drugs in combination substantially inhibited viral reactivation from latency at concentrations that had little or no effect when used singly. These synergistic effects may have implications for viral functions during pathogenesis and for antiviral chemotherapy.

Animals↗
PubMed1995

Mutations that specifically impair the DNA binding activity of the herpes simplex virus protein UL42.

The herpes simplex virus DNA polymerase is a heterodimer consisting of a catalytic subunit and the protein UL42, which functions as a processivity factor. It has been hypothesized that UL42 tethers the catalytic subunit to the DNA template by virtue of DNA binding activity (J. Gottlieb, A. I. Marcy, D. M. Coen, and M. D. Challberg, J. Virol. 64:5976-5987, 1990). Relevant to this hypothesis, we identified two linker insertion mutants of UL42 that were unable to bind to a double-stranded-DNA-cellulose column but retained their ability to bind the catalytic subunit. These mutants were severely impaired in the stimulation of long-chain-DNA synthesis by the catalytic subunit in vitro. In transfected cells, the expressed mutant proteins localized to the nucleus but were nonetheless deficient in complementing the growth of a UL42 null virus. Thus, unlike many other processivity factors, UL42 appears to require an intrinsic DNA binding activity for its function both in vitro and in infected cells. Possible mechanisms for the activity of UL42 and its potential as a drug target are discussed.

Animals↗
PubMed1995

Initiator elements and regulated expression of the herpes simplex virus thymidine kinase gene.

Regulated expression of thymidine kinase mRNA in herpes simplex virus mutants harboring thymidine kinase promoters that lacked functional TATA boxes was largely unaffected by additional sequence alterations around the transcriptional start site. A strong initiator element increased the regulated expression of a TATA-containing promoter by 50% but did not affect that of the TATA-less promoter. Thus, initiator elements exert only small effects in this promoter context.

Animals↗
PubMed1995

Quantification of transcripts from the ICP4 and thymidine kinase genes in mouse ganglia latently infected with herpes simplex virus.

Herpes simplex virus establishes latency in nervous tissue in which it is maintained for the life of the mammalian host, with occasional reactivation leading to subsequent spread. Latency-associated transcripts are abundant during latency, but viral proteins and productive cycle RNAs have not been detected. Using sensitive, quantitative PCR assays, we have quantified certain viral RNAs specific to productive-cycle genes in mouse ganglia latently infected with herpes simplex virus type 1. Sense-strand RNA specific to the essential immediate-early gene, ICP4, was present in most ganglia in variable amounts relative to the amount of viral DNA, with one to seven molecules of RNA per viral genome in about 20% of ganglia. In contrast, the amount of latency-associated transcripts was much less variable, at an average of 4 x 10(4) molecules per viral genome. The amounts of ICP4-specific RNA were similar at 30 and 60 days postinfection, and at least some of these transcripts initiated within a region consistent with utilization of the ICP4 promoter. RNA specific to the thymidine kinase gene, whose transcription in productive infection is dependent on ICP4, was present in latently infected ganglia at a maximum level of 3.2 x 10(6) molecules per ganglion (500 molecules per viral genome). ICP4-specific and tk-specific RNAs measured from the same samples showed a positive correlation extending over 2 orders of magnitude. We conclude that ICP4-specific RNA is expressed in the absence of detectable reactivation and discuss possible implications of our findings for latent gene expression.

Animals↗
PubMed1995

Induction of transcription by a viral regulatory protein depends on the relative strengths of functional TATA boxes.

The mechanisms by which viral regulatory proteins activate the cellular transcription apparatus without binding to specific DNA elements are not fully understood. Several lines of evidence suggest that activation by one such regulatory protein, herpes simplex virus ICP4, could be mediated, at least in part, by TFIID. To test this model, we replaced the TATA box of the ICP4-responsive viral thymidine kinase gene with functional TATA boxes that displayed different apparent affinities for TATA-box-binding protein as measured by DNase I footprinting. We measured the effects of these TATA boxes on ICP4 induction by constructing ICP4-deficient recombinant viruses containing the different TATA alleles and comparing their expression in cells lacking or expressing ICP4. Overall, ICP4 induced weak TATA boxes (those that displayed low apparent affinity for TATA-box-binding protein and low basal expression) the most (18- to 41-fold) and strong TATA boxes the least (7- to 10-fold). Therefore, ICP4 induction correlated inversely with TATA box strength. Using a reconstituted in vitro transcription assay, we determined that the relative levels of induction by ICP4 of the different TATA alleles were similar to those measured in vivo, suggesting that ICP4 was the only viral protein required for induction. These results fit a model in which ICP4 acts in part to enhance binding of TFIID to the TATA box. We compare and contrast these results with those observed with the viral regulatory proteins adenovirus E1a and simian virus 40 large T antigen and the cellular coactivator PC4.

Base Sequence↗