Biomedical subjects

E K Wagner

Publications and source records attributed to E K Wagner.

At least 55 records · Page 3Linked to original sources

PubMed1989

Transcriptional activation is not responsible for increased levels of autonomously expressed simian virus 40 T-antigen in herpes simplex virus-infected cells.

Herpes simplex virus type 1 (HSV-1) superinfection of CV-1 cells weakly transactivated a plasmid-borne metallothionein 1 (MT-1) promoter, but activated the expression of a marker gene controlled by an authentic HSV-1 promoter to a high level. In contrast, CMT-3 cells, which are CV-1 cells stably transformed with the simian virus 40 (SV40) large T-antigen (T-Ag) gene controlled by the MT-1 promoter, contained high levels of T-Ag following HSV-1 superinfection, but only if cells were preincubated in the presence of heavy-metal ions. This T-Ag was functional in that it could mediate the increase in copy number of a marker plasmid containing the SV40 origin of DNA replication. Pulse and continuous labeling of preinduced CMT-3 cells showed that T-Ag expression was not induced by HSV-1; but rather, HSV-1 superinfection resulted in the stabilization of pre-existing protein.

Animals↗

PubMed1989

Transcriptional activation with concurrent or nonconcurrent template replication has differential effects on transient expression from herpes simplex virus promoters.

We have used two methods to induce template replication in order to assess the effect on expression of marker genes controlled by herpes simplex virus type 1 (HSV-1) promoters. One method used the HSV-1 origin of DNA replication from the short repeat region of the viral genome (HSV-1 oris), and allowed simultaneous replication and transcriptional activation of the plasmid-borne template. The other, using the simian virus 40 origin of replication (SV40 ori) allowed plasmid template replication prior to activation of transcription by HSV-1 infection. The two regimes had markedly different effects upon the levels of reporter gene activity induced by HSV-1 superinfection. Replication of reporter plasmids using the SV40 ori yielded levels of reporter gene activity proportional to plasmid copy number when cells were superinfected with HSV-1. In contrast, our results indicated that sequences containing, or in close proximity to, the HSV-1 oris in the reporter plasmid had a significant inhibitory effect on expression from all viral promoters whether or not the plasmid was allowed to replicate. Still, the early (beta) promoter-controlled reporter enzyme activity declined at late times while that controlled by the strict late (gamma) promoter was significantly higher following HSV-1 oris-mediated template replication.

Animals↗

PubMed1989

Molecular and biological characterization of a type 1 herpes simplex virus (HSV-1) specifically deleted for expression of the latency-associated transcript (LAT).

We have previously reported that in sensory neurons of mice harboring latent herpes simplex virus (HSV), a novel virus-encoded RNA species termed the latency-associated transcript (LAT) is abundant. To investigate a potential function for LAT in the latent state, we have engineered a deletion in HSV-1 strain KOS(M) which abolishes LAT expression. The deletion virus (KOS 8117) is not altered for virulence or the ability to replicate in mouse cells in vivo or in vitro. Although no transcripts were detected in lumbosacral ganglia of mice after virus inoculation on scarified rear footpads, the deletion virus established a latent infection since infectious virus could be recovered by explanting and cultivating ganglia in vitro with permissive cells. These results indicate that expression of LAT is not an absolute requirement for the establishment or maintenance of the latent state or for recovery of infectious virus from the latent state by in vitro co-cultivation. Since no viral-encoded transcripts were detected in cells latently infected with KOS 8117, maintenance of the latent infection also appears to be exclusively a neuronal function.

Animals↗

PubMed1989

Herpes simplex virus type 1 latency-associated transcription plays no role in establishment or maintenance of a latent infection in murine sensory neurons.

Using herpes simplex viruses deleted and restored for the latency-associated transcripts (LATs), we have quantitatively assessed the role of the transcripts in establishment and maintenance of latent infection. Determination of the number of neurons latently infected and the copy number of viral genomes per latently infected ganglion indicated that there is no difference between viruses expressing and not expressing the transcripts. In addition, the amount of viral DNA present in ganglia latently infected with the LAT-negative virus KOS 8117 did not differ from the value for LAT+ counterparts over an 11-month period of analysis. From these results we conclude that LATs play no role in establishment or maintenance of a latent infection with herpes simplex virus type 1. If these transcripts play a role in latency, they must function during the reactivation step.

Animals↗

PubMed1989

Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species in latently infected mouse neurons. Previous sequence analysis revealed no obvious promoter elements near the 5' end of the LAT, but a TATA box and other potential promoter elements were found 700 base pairs upstream. A recombinant virus in which the rabbit beta-globin gene was inserted immediately downstream of the TATA box expressed globin mRNA and did not express the LAT. A second recombinant virus, in which this TATA box was removed, was negative for LAT expression in a latent infection. The location of the LAT promoter suggested that RNA upstream of the LAT was synthesized and degraded during latent-phase transcription. Low levels of this RNA were observed by in situ hybridization. In other experiments, RNA from a productive infection was used to detect a transcript extending from the LAT promoter to a polyadenylation signal approximately 8.5 kilobase downstream. These data suggest that the LAT may be processed from a larger transcription unit which begins distal to the TATA box 700 base pairs upstream of the LAT and extends to a polyadenylation signal almost 5 kilobases downstream of the 3' end of the LAT.

Animals↗

PubMed1988

Partial rescue of herpes simplex virus neurovirulence with a 3.2 kb cloned DNA fragment.

A herpes simplex virus (HSV) intertypic recombinant (RE6) has been previously shown to be non-neurovirulent following direct intracranial inoculation of mice. An in vitro recombination/in vivo selection strategy was employed to further characterize the gene or genes responsible for the avirulence of this mutant. It was found that RE6 could be converted to a lethal virus by incorporation of wild-type HSV-1 sequences mapping between 0.698 and 0.721 map units. Restriction endonuclease and Southern blot analysis revealed that viral recombinants which incorporated at least part of the cloned 17syn+ sequences were selected by passage in mouse brains in vivo. The recombinants generated with this fragment were at least 50-fold more neuroviurlent than RE6, as determined by the plaque forming unit to lethal dose 50% assay. Further, they displayed a significant increase in ability to replicate in mouse brain tissue following intracranial inoculation. However, these recombinants did not display a replication advantage over RE6 in cultured mouse cells at 38.5 degrees C. Thus, the defect present within this region of the RE6 genome specifically affects replication in the nervous system. In the accompanying paper we analyze the RNA transcription and DNA sequence in this portion of the RE6 and parental strain genomes.

Cloning, Molecular↗

PubMed1988

DNA sequence and RNA transcription through a site of recombination in a non-neurovirulent herpes simplex virus intertypic recombinant.

RE6 is a herpes simplex type-1 (HSV-1) X herpes simplex type-2 (HSV-2) intertypic recombinant that cannot replicate in the adult mouse nervous system. In the accompanying report, we have shown that HSV-1 sequences between 0.698 and 0.721 map units can restore a partial neurovirulent phenotype to RE6. In this report, we have used comparative DNA sequence analysis of RE6, 17syn+ (HSV-1) and HG52 (HSV-2) to demonstrate that this region contains a site of recombination between HSV-1 and HSV-2 sequences in RE6. High resolution transcription analysis has demonstrated that three readily detected transcripts are present in this region of the genome. In addition, the 5' end of a low abundance 5 kb transcript was also located in the right-hand portion of this region. All the transcripts encoded by HSV-1 and HSV-2 in this region of the genome are expressed by the RE6 recombinant. This and our sequence data suggest that the lack of neurovirulence in RE6 is not due to a simple loss in the expression of a transcript or to a defect in a protein encoded by a gene at the site of recombination.

Base Sequence↗

PubMed1988

A herpes simplex virus transcript abundant in latently infected neurons is dispensable for establishment of the latent state.

We have previously reported that a novel herpes simplex virus RNA transcript partially overlapping the gene encoding ICPO and expressed from the opposite DNA strand is abundant in sensory neurons of mice harboring a latent infection [J.G. Stevens, E.K. Wagner, G.B. Devi-Rao, M.L. Cook, and L.T. Feldman, Science 235, 1056-1059 (1987)]. This finding suggested that this transcript might be involved in establishment, maintenance, or reactivation of latent virus. To determine the function of this latency-associated transcript (LAT), we have examined the latency characteristics of a deletion mutant which is unable to express the LAT gene. Although no viral transcripts could be found in the lumbosacral ganglia of mice surviving rear footpad infection with this deletion virus, a latent infection had been established since infectious virus could be induced and detected after explanation and cocultivation of ganglia with permissive cells in culture. These results indicate that HSV-1 LAT expression is not an absolute requirement for establishment of the latent state.

Animals↗

PubMed1988

Prominence of the herpes simplex virus latency-associated transcript in trigeminal ganglia from seropositive humans.

Although herpes simplex virus type 1 (HSV-1) is known to reside latently in trigeminal ganglia between episodes of reactivation, the mechanisms involved in restricting the virus to this state are not understood. Using in situ nucleic acid hybridization methods, we show that there is HSV-encoded RNA in ganglion cells from 10 of 12 seropositive and zero of three seronegative patients studied at autopsy. Transcripts mapping to the region encoding the immediate-early polypeptide ICPO and the latency-associated transcript (LAT) were detected in the nuclei of these neurons. No other region of the HSV-1 genome was found to be expressed. When carefully defined probes were used to identify the transcripts, RNA corresponding to the LAT and, rarely, to ICPO was found. These results suggest that HSV-1 latency is an active process and that the LAT may be involved in regulating viral genetic expression.

Herpes Simplex↗

PubMed1988

The herpes simplex virus latency-associated transcript is spliced during the latent phase of infection.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species 2,100 to 2,200 bases in length and a less abundant one ca. 730 bases shorter in latently infected mouse and rabbit neurons. RNA blot hybridization experiments using 20- to 22-base synthetic oligonucleotides and mung bean nuclease protection assays have demonstrated that the smaller LAT species is colinear with the larger one, except for a 730-base intron. On the basis of Northern blot analysis, the spliced species which comprises as much as 50% of the total LAT in latent infections of mice with several strains of herpes simplex virus type 1 and latent infections of rabbits with either the McKrae or the KOS(M) strains of virus is not present in the acute phase of infection. Further and rather surprisingly, in mice latently infected with the KOS(M) strain of virus, the spliced LAT species is considerably less abundant. This suggests that both the strain of virus and the animal in which the latent infection occurs are important in either the processing or stability of spliced LAT. Finally, an exhaustive series of experiments failed to provide convincing evidence that a unique, poly(A)+ species of LAT exists in the latent phase of infection.

Acute Disease↗

PubMed1988

Physical characterization of the herpes simplex virus latency-associated transcript in neurons.

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.

Amino Acid Sequence↗

PubMed1987

RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons.

In initial attempts to define the molecular events responsible for the latent state of herpes simplex virus, in situ hybridization was utilized to search for virally encoded RNA transcripts in latently infected sensory neurons. The use of cloned probes representing the entire viral genome indicated that transcripts encoded within terminal repeats were present. When the alpha genes encoding ICP-0, ICP-4, and ICP-27 and the gamma 1 gene encoding VP-5 were employed, only RNA transcripts hybridizing to the ICP-0 probe were detected. In latently infected cells, the ICP-0--related transcripts were localized principally in the nucleus; this was not the case in acutely (productively) infected neurons or in neurons probed for RNA transcripts coding for actin. In Northern blotting experiments, an RNA of 2.6 kilobases was detected with the ICP-0 probe. When single-stranded DNAs from the ICP-0 region were used as probes, RNA from the strand complementary to that encoding ICP-0 messenger RNA (mRNA) was the major species detected. This RNA species may play a significant role in maintaining the latent infection.

Animals↗

PubMed1987

The kinetics of expression of individual herpes simplex virus type 1 transcripts.

We have used specific single-stranded DNA probes cloned in M13 to analyze the kinetics of expression of 20 individual herpes simplex virus type 1 (HSV-1) transcripts, most of known function. The three alpha transcripts studied all differ in the details of their shut-off. There are at least three groups of beta or early transcripts which differ both in the time of their maximum rate of synthesis and in the time of their shut off. Similarly late (beta gamma and gamma) transcripts form a complex group all of which share the fact that their rates of synthesis reach a maximum at a time when beta transcripts are being shut off.

Animals↗

PubMed1987

A bi-functional reporter plasmid for the simultaneous transient expression assay of two herpes simplex virus promoters.

We have constructed a transient expression vector (pCAL) containing two reporter genes, chloramphenicol acetyltransferase (CAT) and beta-galactosidase (beta-gal) for use in studying herpes simplex virus type 1 (HSV-1) promoter activity in mammalian cells. The construct was designed to be useful in analyzing the simultaneous expression from two different promoters. To test the utility of the vector, we used three HSV-1 promoters that had been characterized previously by workers in this laboratory. Two are early (beta) promoters, for alkaline exonuclease and deoxyuridine triphosphate nucleotidohydrolase; the third promoter controls the major capsid protein transcript and is late (beta gamma). The two different kinetic classes of promoters were ligated in a divergent orientation into pCAL and transfected into rabbit skin fibroblast. Transfected cells were then superinfected with low multiplicities of HSV-1; 18 hr later, we observed the simultaneous expression of both marker genes under control of the respective promoters. The usefulness of such a transient expression reporter vector is discussed.

Animals↗

PubMed1987

The effect of elevated levels of herpes simplex virus alpha-gene products on the expression of model early and late genes in vivo.

The rate of synthesis in vivo and the steady-state level of mRNA of four "model" herpes simplex virus type 1 (HSV-1) genes were measured as a function of high levels of alpha-gene products. The genes studied were ICP4 (alpha), deoxy-UTPase (beta), VP5 (beta gamma), and glycoprotein C (gC, gamma). Accumulation of high levels of alpha proteins was accomplished either by infection with an HSV-1 mutant, temperature-sensitive in ICP4 (ts606) at the nonpermissive temperature then shift-down to permissive temperature, or by infection with wild-type virus under cycloheximide blockage of protein synthesis followed by release. Compared to RNA expression in normal infections, beta gamma and gamma transcription rates were both transiently stimulated under the experimental conditions employed. The greatest effect was seen with the gamma-gC mRNA transcription rates. In addition, at nonpermissive temperatures with ts 606, the amount of expression of gC mRNA was significantly increased over normal early levels, in contrast to the case with the VP5 transcript. The impact of such results on models of HSV gene expression in vivo are discussed.

Animals↗

PubMed1987

Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation.

Acetyltransferases↗

PubMed1986

Characterization of the genes encoding herpes simplex virus type 1 and type 2 alkaline exonucleases and overlapping proteins.

A detailed sequence analysis of the herpes simplex virus type 1 (HSV-1) and HSV-2 DNA encoding the alkaline exonuclease mRNA clusters has been completed. Three partially colinear mRNAs (2.3, 1.9, and 0.9 kilobases) are completely encoded within the DNA sequence presented. The putative promoter regions of the transcripts were inserted upstream of a plasmid-borne chloramphenicol acetyl transferase (CAT) gene and assayed for their ability to induce transcription of the CAT gene upon low multiplicity of infection with HSV in transient expression assays. We conclude that the expression of all three transcripts appear to be controlled by individual promoters. The 2.3-kilobase mRNA contains an open translational reading frame sufficient to encode 626 amino acids for the HSV-1 alkaline exonuclease enzyme; this value is 620 amino acids for HSV-2. A comparison of the predicted amino acid sequences of the HSV-1 and HSV-2 alkaline exonuclease enzymes revealed significant amino acid differences in the N-terminal portions of the two proteins; however, computer analyses suggest that the three-dimensional structures of the HSV-1 and HSV-2 nuclease enzymes are very similar. The 0.9-kilobase mRNA contains an open reading frame which shares a small amount of out-of-phase overlap with the C-terminal portion of the alkaline nuclease open reading frame. This open reading frame has the capacity to encode a 96-amino-acid polypeptide (10,500 daltons).

Acetyltransferases↗

PubMed1986

Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS.

It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness.

Animals↗