Search PubMed⌕ Search

Biomedical subjects

E K Wagner

Publications and source records attributed to E K Wagner.

At least 37 records · Page 2Linked to original sources
PubMed1996

A 348-base-pair region in the latency-associated transcript facilitates herpes simplex virus type 1 reactivation.

Latency-associated transcript (LAT) promoter deletion mutants of herpes simplex virus type 1 have a reduced capacity to reactivate following adrenergic induction in the rabbit eye model. We have mapped a reactivation phenotype within LAT and describe the construction of recombinants in which poly(A) addition sites have been placed at intervals within the LAT region to form truncated LAT transcripts. These mutants localize the induced reactivation phenotype to the 5' end of LAT. To further define this region, we constructed a recombinant containing a 348-bp deletion located 217 bp downstream of the transcription start site of the 8.5-kb LAT. This virus, 17delta348, expresses LAT but exhibits a significantly reduced ability to reactivate following epinephrine iontophoresis into the cornea. Quantitative DNA PCR analysis reveals that 17delta 348 establishes a latent infection within rabbit trigeminal ganglia with the same efficiency as does either the rescuant or wild-type virus. The region deleted in 17delta348 encodes three potential translational initiators (ATGs) which we have mutated and demonstrated to be dispensable for epinephrine-induced reactivation. In addition, three smaller deletions within this region have been constructed and were shown to reactivate at wild-type (parent) frequencies. These studies indicate that an undefined portion of the 348-bp region is required to facilitate induced reactivation. Sequence analysis of this 348-bp region revealed a CpG island which extends into the LAT promoter and which possesses homology to conserved elements within the mouse and human XIST transcript encoded on the X chromosome. Possible implications of these elements in the regulation of LAT expression are discussed.

Animals↗
PubMed1995

Herpes simplex virus recombination vectors designed to allow insertion of modified promoters into transcriptionally "neutral" segments of the viral genome.

The use of recombinant viruses has been essential in investigation of the biology of herpes simplex virus (HSV). In this communication we describe a number of viral recombination vectors that we have generated for use in promoter structure/function analysis within the context of the HSV-1 genome. We have utilized two regions of the HSV genome that contain genes nonessential for replication in cultured cells--the glycoprotein C (gC or UL44) locus in the UL of the genome and the area encompassing the promoter and 5' portion of the latency associated transcript (LAT) within the RL factual influence on promoters due to the site of insertion. Two different kinetic promoters were analyzed, those controlling expression of the gamma UL 38 and the beta dUTPase genes, in both loci. All constructs tested displayed reporter gene mRNA expression with expected kinetics, and we conclude that there are no neighboring cryptic promoter elements that could interfere with expression studies using the vectors described.

Animals↗
PubMed1995

Two overlapping transcription units which extend across the L-S junction of herpes simplex virus type 1.

A region of the herpes simplex virus type 1 genome located upstream of the alpha 0 promoter contains a promoter which regulates transcription in the opposite orientation to that driven by alpha 0. Analyses of mutants from which this promoter, alpha X, was deleted and a mutant in which a fragment that serves as a transcription terminator and polyadenylation signal was inserted upstream of this promoter demonstrate that two distinct transcription units overlap this region of the genome and are transcribed in a direction antisense to the neurovirulence gene gamma (1)34.5. One unit, dependent on the alpha X promoter, is active when cells are infected in the presence of the protein synthesis inhibitor cycloheximide. The second unit, independent of alpha X, is active during the course of productive infection. This transcription unit originates from a promoter upstream of alpha X which is distinct from the latency-associated promoter (LAP). Two polyadenylated transcripts of 0.9 and 4.9 kb accumulate from this region of the genome during productive infection, but no mature transcripts accumulate in infected cells maintained in the presence of cycloheximide. Kinetic analyses demonstrate that the transcripts that accumulate during productive infection fall into the beta class of herpes simplex virus type 1 genes.

Animals↗
PubMed1994

Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor.

The herpes simplex virus (HSV) type 1 strict late (gamma) UL38 promoter contains three cis-acting transcriptional elements: a TATA box, a specific initiator element, and the downstream activation sequence (DAS). DAS is located between positions +20 and +33 within the 5' untranslated leader region and strongly influences transcript levels during productive infection. In this communication, we further characterize DAS and investigate its mechanism of action. DAS function has a strict spacing requirement, and DAS contains an essential 6-bp core element. A similarly positioned element from the gamma gC gene (UL44) has partial DAS function within the UL38 promoter context, and the promoter controlling expression of the gamma US11 transcript contains an identically located element with functional and sequence similarity to UL38 DAS. These data suggest that downstream elements are a common feature of many HSV gamma promoters. Results with recombinant viruses containing modifications of the TATA box or initiator element of the UL38 promoter suggest that DAS functions to increase transcription initiation and not the efficiency of transcription elongation. In vitro transcription assays using uninfected HeLa nuclear extracts show that, as in productive infection with recombinant viruses, the deletion of DAS from the UL38 promoter dramatically decreases RNA expression. Finally, electrophoretic mobility shift assays and UV cross-linking experiments show that DAS DNA forms a specific, stable complex with a cellular protein (the DAS-binding factor) of approximately 35 kDa. These data strongly suggest that the interaction of cellular DAS-binding factor with DAS is required for efficient expression of UL38 and other HSV late genes.

Animals↗
PubMed1994

Herpes simplex virus type 1 DNA replication and gene expression during explant-induced reactivation of latently infected murine sensory ganglia.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus DNA replication and gene expression in two murine in vitro models for virus reactivation. We examined latent infections with wild-type (wt), precisely defined latency-associated transcript-negative (LAT-) mutants, and LAT+ rescuants of these mutants of the 17syn+ strain of virus in both murine trigeminal and lumbosacral ganglia and of the KOS(M) strain in the latter. In explants of ganglia latently infected with the LAT- mutant of strain 17syn+ virus, a reduction in number of cultures exhibiting cytopathic effects due to virus reactivation and measurable delays in virus recovery were observed compared with wt or the LAT+ rescuant. This LAT-specific effect was not seen in explants of lumbosacral ganglia latently infected with mutants derived from the KOS(M) strain of virus. Although there was appreciable variation between individual animals, no significant difference between LAT+ and LAT- virus in time of onset of viral DNA replication in explanted ganglia was seen with use of either virus strain. There was a slight decrease in the relative amount of viral DNA recovered compared with internal cellular controls in latently infected ganglia harboring the LAT- mutant of 17syn+ compared with the wt virus or the LAT+ rescuant. This reduced relative amount ranged from 0 to as much as 50% but averaged 20%. Such differences were not seen in infections with KOS(M)-derived mutants. In contrast, although expression of productive-cycle transcripts could be detected within 4 h following explant cultivation of latently infected ganglia, no differences between LAT+ and LAT- viruses could be seen. As discussed, these data place specific constraints on possible models for the role of LAT expression in in vitro reactivation systems.

Animals↗
PubMed1994

Molecular analysis of herpes simplex virus type 1 during epinephrine-induced reactivation of latently infected rabbits in vivo.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus (HSV) DNA replication and gene expression in the rabbit corneal model for virus reactivation in vivo. We used carefully defined latency-associated transcript-negative (LAT-) and LAT+ promoter mutants of the 17syn+ strain of HSV type 1. In agreement with earlier studies using a more extensive LAT- deletion mutant, the 17 delta Pst(LAT-) virus reactivated with extremely low frequency upon epinephrine induction. In contrast to our findings with murine latency models, amounts of viral DNA recovered from rabbit ganglia latently infected with either LAT+ or LAT- virus were equivalent. Also in contrast with the murine models, no net increase in viral DNA was seen in latently infected rabbit trigeminal ganglia induced to reactivate in vivo by iontophoresis of epinephrine. Despite this, transcription of lytic-phase genes could be detected within 4 h following induction of rabbits latently infected with either LAT+ or LAT- virus; this transcription diminished by 16 h following induction. These results are discussed in relation to models for the mechanism of action of HSV LAT.

Animals↗
PubMed1994

The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected.

As do many other alphaherpesviruses, pseudorabies virus (PRV) transcribes a limited portion of its viral genome in latently infected neurons during latency. The sequence of the PRV latency-associated transcript (LAT) is bounded on its 5' end by a putative promoter region which contains sequence elements similar to those characterized for the herpes simplex virus (HSV) LAT promoter. Using the bacterial beta-galactosidase gene as a reporter, we have assayed PRV LAT promoter activity in the genomic environment in recombinant HSVs. The PRV LAT promoter-beta-galactosidase reporter gene was recombined into the terminal and internal long repeat regions (RL regions), replacing the normal HSV LAT promoter, the cap site, and the first 60 bases of the primary transcript. When recombined into the RL region, appreciable reporter gene expression was observed following infection of two cell lines of neuronal origin; little or no activity was seen with these recombinants following infection of rabbit skin or mouse embryo fibroblasts. No significant expression was seen when the promoter was recombined into the gC locus in the long unique region in any of the cell types utilized. Such results suggest that the PRV latency promoter contains neuronal cell-specific elements and that the HSV RL region provides an appropriate genomic environment for the manifestation of that specificity.

Base Sequence↗
PubMed1994

The herpes simplex virus type 1 major capsid protein (VP5-UL19) promoter contains two cis-acting elements influencing late expression.

The herpes simplex virus type 1 (HSV-1) major capsid protein VP5 gene (UL19) is expressed with beta gamma (gamma 1 [leaky late]) kinetics. We have previously described the construction of recombinant HSV-1 in which the VP5 promoter was engineered to control the expression of the bacterial beta-galactosidase gene as a reporter (C.-J. Huang, S. A. Goodart, M. K. Rice, J. F. Guzowski, and E. K. Wagner, J. Virol. 67:5109-5116, 1993). Here we describe further mutational analysis in recombinant viruses. We have precisely defined the boundaries of the VP5 promoter and identified two regions important for both the level and the kinetics of expression. The 5' boundary was located at -48 relative to the initiation site of transcription by analyzing a series of nested deletions in the upstream sequence, and although a number of cis-acting sites influencing transient expression have been identified upstream of this point, these sites have no role in promoter activity during productive infection. Deletion of an Sp1-binding site located between -48 and the TATA box at -30 greatly reduced VP5 promoter activity late but not early after infection. A cis-acting element whose sequence resembles the human immunodeficiency virus type 1 initiator was located between -2 and +10 in the VP5 sequence by characterizing a series of deletions and site-directed block mutations downstream the TATA box. This element defines the 3' limit of the VP5 promoter, and like the upstream element, disruption of this element also inhibited promoter activity late in the productive cycle.

Base Sequence↗
PubMed1993

Transcriptional analysis of the herpes simplex virus type 1 region containing the TRL/UL junction.

In this study we have used a combination of Northern blot, cDNA, and RNAse protection analysis to characterize transcription occurring at the junction of the long terminal repeat (TRL) and long unique segment (UL) of herpes simplex virus type 1 (HSV-1). Two low abundance 5' colinear transcripts mapped between the junction of the TRL/UL and the cap site of the primary latency associated transcripts (LAT). Analysis of the region containing the first four open translational reading frames (ORFs) of the UL segment of the virus revealed a complex pattern of overlapping transcripts and polyadenylation site utilization. Inefficient termination late, but not early in infection, at the polyadenylation site following the UL2 ORF leads to an unusual situation where the promoter controlling a beta gamma transcript encoding the UL1 ORF also controls a 5' colinear gamma transcript. Further, although the entire UL3 ORF is conserved between HSV-1 and HSV-2, the only abundant uniquely UL3 encoding transcript in both virus types was found to originate within the UL3 ORF and has the capacity to encode only a truncated UL3 protein product.

Animals↗
PubMed1993

The herpes simplex virus type 1 reactivation function lies outside the latency-associated transcript open reading frame ORF-2.

The latency-associated transcription unit has been shown to be important for in vivo reactivation of herpes simplex virus from the latent state. A recombinant virus was constructed to alter the largest open reading frame in this region. This virus had a wild-type reactivation phenotype, suggesting that herpes simplex virus does not require a protein function from this reading frame for efficient reactivation from latency.

DNA, Recombinant↗
PubMed1993

Mutational analysis of the herpes simplex virus type 1 strict late UL38 promoter/leader reveals two regions critical in transcriptional regulation.

The unusual TATA homology TTTAAA at -31 relative to the transcriptional start site of the herpes simplex virus type 1 (HSV-1) strict late (gamma) UL38 gene defines the 5' extent of this promoter in recombinant virus. We have further analyzed this promoter by generating recombinant viruses containing nested deletions 3' of the transcriptional start site and with recombinant viruses containing specific promoter/leader alterations. A recombinant virus containing the UL38 promoter/leader from -50 to +9 expressed reporter gene enzyme levels at approximately 10% of those from a recombinant containing the full viral promoter/leader (-50 to +99). The accumulation of reporter gene mRNA in infections with the -50 to +9 recombinant was still regulated with gamma kinetics. Further removal of UL38 leader sequences resulted in a nearly complete loss of expression. Analysis of promoter chimera recombinant viruses has shown that sequences downstream of the TATA box and spanning the transcriptional start site of the UL38 promoter are functionally distinct from those of either the beta UL37 gene or the beta gamma VP16 (UL48) gene; thus, we conclude that sequences from -31 to +9 of the UL38 gene constitute a core gamma promoter. Further deletional and substitutional analyses have also demonstrated the presence of a 14-bp element (the downstream activation sequence) located between +20 to +33 in the nontranslated leader region which is required for full levels of transcription.

Animals↗
PubMed1993

Mutational analysis of sequences downstream of the TATA box of the herpes simplex virus type 1 major capsid protein (VP5/UL19) promoter.

Transient expression assays with the herpes simplex virus type 1 (HSV-1) promoter/leader controlling the beta gamma (leaky-late) VP5 (UL19) mRNA encoding the major capsid protein showed that no more than 36 to 72 bases of VP5 leader are required for full-level expression. Constructs lacking the viral leader and the transcription initiation site expressed the reporter gene at about 20% of the maximum level. We confirmed this observation by using recombinant viruses in which VP5 promoter/leader deletions controlling the bacterial beta-galactosidase gene were inserted into the nonessential glycoprotein C (UL44) locus of the genome. Sequences within +36 are required for full-level expression, and removal of all leader sequences including the cap site resulted in a 10-fold decrease in reporter mRNA accumulation. The removal of the leader sequence had a measurable effect upon the kinetics of reporter mRNA accumulation, but insertion of the entire VP5 leader and cap site into a construct in which the reporter gene was controlled by the kinetically early (beta) dUTPase (UL50) promoter did not result in any significant change in the kinetics of dUTPase promoter expression. These results suggest that DNA sequences both 5' and 3' of the TATA box are important determinants of the beta gamma kinetics and levels of VP5 mRNA accumulation in the infected cell.

Animals↗
PubMed1992

Effect of genomic location on expression of beta-galactosidase mRNA controlled by the herpes simplex virus type 1 UL38 promoter.

To examine the effect of genomic location on the details of expression of selected herpes simplex virus promoters, we have constructed recombination vectors for placing such promoters controlling the beta-galactosidase reporter gene into two regions of the viral genome lacking any nearby promoter or regulatory elements. The first vector generates the promoter-beta-galactosidase reporter gene inverted within the locus of the gC (UL44) translational reading frame; the second replaces the LAT promoter and the first 600 bases of the primary transcript in both copies of the RL region. These locations were chosen to obviate any possible influence of upstream but noncontiguous heterologous or homologous DNA sequence elements upon promoter activity. When the reporter gene controlled by the strict late (gamma) UL38 promoter was placed in the gC location, it was significantly less active than in its normal location; in contrast, promoter activity was comparable to wild-type values when the promoter was recombined into the RL region. The low level of activity in the gC location could be partially alleviated by the incorporation of additional DNA sequences upstream of the UL38 promoter. Despite the effect of genomic location upon the level of expression, the kinetics of expression in either location mirrors the wild-type UL38 strict late kinetics of expression. Finally, we used deletional analysis to demonstrate that no more than 29 bases of DNA sequence 5' of the mRNA cap site are required for promoter activity in either location; this result is consistent with earlier results of transient-expression assays and indicates that the UL38 promoter shares general features with other strict late (gamma) herpes simplex virus promoters.

Animals↗
PubMed1991

Analysis of the herpes simplex virus type 1 promoter controlling the expression of UL38, a true late gene involved in capsid assembly.

The cistrons encoding the herpes simplex virus type 1 (HSV-1) UL37 and UL38 genes are adjacent to one another but are transcribed from opposite strands of the viral DNA. The UL37 gene encodes a 1,123-amino-acid protein of unknown function, while the 465-amino-acid UL38 protein is involved in capsid assembly. Previous work from our laboratory indicated that the transcripts encoding these proteins are expressed with significantly different kinetics in productive infection. In the present communication we confirm the kinetic classes and precisely map the cap sites of the UL37 and UL38 mRNAs. A bifunctional reporter gene vector was used to demonstrate that divergent promoters control the expression of these reporter genes in trans-activation assays. The UL38 promoter is functionally separable from that controlling UL37 in a recombinant virus. We used deletion analysis to demonstrate that as few as 29 bases 5' of the mRNA cap site are adequate for full activity of the UL38 promoter in trans-activation assays. Finally, we analyzed the protein-binding properties of the UL38 promoter; several sites that form complexes containing ICP4, with clear homology to those identified in the HSV-1 gamma 42 promoter, are present. Thus, in general, the properties of this promoter are quite similar to those of other gamma promoters.

Animals↗
PubMed1991

Relationship between polyadenylated and nonpolyadenylated herpes simplex virus type 1 latency-associated transcripts.

RNA from the region of the genome encoding herpes simplex virus type 1 latency-associated transcripts (LATs) expressed during lytic infection yields low abundances of both polyadenylated and nonpolyadenylated forms. As has been previously shown for latent infection (A. T. Dobson, F. Sedarati, G. Devi-Rao, W. M. Flanagan, M. J. Farrell, J. G. Stevens, E. K. Wagner, and L. T. Feldman. J. Virol. 63:3844-3851, 1989), all lytic-phase expression of such transcripts requires promoter elements situated approximately 600 bases 5' of the previously mapped 5' end of the poly(A)- forms of LAT. Transient expression experiments revealed no other clear promoter elements within this region, and relatively small amounts of latent-phase transcripts initiating at the same site as observed for lytic-phase LAT could be detected by RNase protection assays. In the lytic phase of infection, the most abundant forms of polyadenylated LAT extended 1,600 bases from the initiation site near the LAT promoter to a potential splice donor site. Poly(A)- LAT species were not recovered in significant amounts from lytically infected neuroblastoma cells, but such RNA from lytically infected rabbit skin cells comapped with poly(A)- LAT from latently infected sensory neurons. Both map between canonical 5' splice donor and 3' splice acceptor site 1,950 bases apart. Poly(A)- LAT cochromatographed with uncapped rRNA on m-aminophenyl boronate agarose under conditions in which capped mRNA was bound. All of these data confirm the previously presented scheme for the expression of poly(A)- LAT as a stable intron derived from the splicing of a large primary transcript; however, we were unable to detect the spliced polyadenylated product of this splicing reaction.

Animals↗
PubMed1990

Herpes simplex virus latent phase transcription facilitates in vivo reactivation.

The biological role of latent phase transcripts was studied in a rabbit model of herpes simplex virus type I (HSV-I) ocular reactivation. Virus X10-13, a variant of HSV which does not express latency associated transcripts (LAT), has been previously shown to establish latent infection in mouse sensory nerve ganglia, and LAT(-) virus can be recovered upon explantation and cocultivation of ganglia. In the rabbit, we show here that this virus replicates normally on the cornea and conjunctiva and establishes latent infections in corresponding trigeminal ganglionic neurons. However, X10-13 is not efficiently reactivated after iontophoresis of 0.01% epinephrine into the cornea. In contrast, XC-20, a LAT(+) derivative of X10-13 in which LAT expression had been restored by marker rescue of X10-13 with a cloned HSV-I EcoRI J + K fragment, reactivated at a significantly higher rate. Control experiments indicated that XC-20 and X10-13 established latent infections in an equivalent number of neurons. We conclude that latent phase transcription of HSV facilitates ganglionic reactivation and subsequent ocular shedding of the reactivated virus.

Acute Disease↗
PubMed1990

A major portion of the latent pseudorabies virus genome is transcribed in trigeminal ganglia of pigs.

Pseudorabies virus (PRV) is a porcine herpesvirus that establishes latent infections in trigeminal ganglia. To determine whether PRV expresses any transcripts that could play a role in latency, the trigeminal ganglia of 14 pigs previously inoculated through the nose and latently infected with PRV(Ka) were assayed by in situ nucleic acid hybridization for the presence of PRV-specific RNA. Hybridizations employing probes encompassing the entire viral genome revealed that an area extending from 0.64 to 0.82 map units was transcriptionally active. The DNA probe that most consistently detected transcripts was BamHI-8, a fragment which contains the gene for the immediate-early protein. With this probe, ganglia from 10 (71%) of 14 pigs scored positive for PRV RNA, although only 1 (8%) of 12 of the ganglia from the opposite side reactivated virus after explanation and culture of latently infected trigeminal ganglia. The RNA was transcribed from the strand opposite to that coding for the immediate-early protein; the signal was neuronally localized, with dense nuclear accumulation accompanied by variable numbers of grains over the cytoplasm. Northern RNA blot analysis showed that a discrete set of poly(A)- PRV transcripts were present in latently infected trigeminal ganglia. Additional in situ nucleic acid hybridization analysis revealed that the 3' limit of the transcriptionally active area was located in a 1.2-kilobase fragment upstream and adjacent to the 5' end of the immediate-early protein RNA, whereas the 5' limit was as much as 4.9 kilobases downstream from the 3' end of this RNA. PRV therefore expresses latent-phase transcripts that, although similar in many respects to latent-phase transcripts reported for other herpesviruses, have some unique properties.

Animals↗