ABSTRACT
During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.
Subject(s)
DNA-Binding Proteins/biosynthesis , Herpesvirus 1, Human/physiology , Immediate-Early Proteins , Promoter Regions, Genetic , Transcription Factors/biosynthesis , Transcription, Genetic , Virus Latency , Animals , Base Sequence , Binding Sites , Chloramphenicol O-Acetyltransferase/biosynthesis , Consensus Sequence , Cytomegalovirus/genetics , DNA Primers , DNA-Binding Proteins/metabolism , Early Growth Response Protein 1 , Genes, Reporter , Herpesvirus 1, Human/genetics , Humans , Mice , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , TATA Box , Transcription Factors/metabolism , Virus Activation , Zinc FingersABSTRACT
Clinical isolates of human cytomegalovirus (HCMV) were screened for susceptibility to ganciclovir by plaque-reduction assay and in situ ELISA. A pretreatment isolate of HCMV obtained from the bronchial brushing of a heart transplant recipient contained both ganciclovir-susceptible and -resistant virus. Ganciclovir-susceptible (P8) and -resistant (D16) strains were further isolated by plaque purification. Both strains phosphorylated ganciclovir at levels similar to the ganciclovir-susceptible strain AD169. D16 was also resistant to phosphonoformic acid and to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and cytosine. These data suggest that the resistance of D16 to these drugs results from a mutation in the viral DNA polymerase gene.
Subject(s)
Cytomegalovirus/drug effects , Ganciclovir/pharmacology , Nucleic Acid Synthesis Inhibitors , Antiviral Agents/pharmacology , Chromatography, High Pressure Liquid , Cytomegalovirus/growth & development , Drug Resistance, Microbial , Enzyme-Linked Immunosorbent Assay/methods , Foscarnet , Humans , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/pharmacology , Viral Plaque Assay/methodsABSTRACT
An in situ ELISA was developed as an improved procedure over the plaque reduction assay for antiviral susceptibility testing of HCMV. Unlike the plaque reduction assay, the ELISA can be completed at 4-5 days post-infection. The effective dose (ED50) of ganciclovir (GCV), acyclovir (ACV), phosphonoacetic acid (PAA), or phosphonoformic acid (PFA), was determined using HCMV strain AD169. The resistance profiles of two laboratory-derived GCV-resistant mutants of HCMV strain AD169 and seven clinical isolates were determined using the ELISA. The ELISA results were confirmed by the plaque reduction assay. The ED50 for GCV with the AD169 control ranged from 3.1 to 6.2 microM with a mean inhibitory concentration of 5.4 +/- 1.4 microM. Six of the clinical isolates were susceptible to GCV (ED50 = 3.1-6.2 microM). The seventh isolate had an ED50 of 50 microM and was resistant to GCV. This ELISA assay is reproducible and relatively simple to perform. The ELISA endpoints are clearly determined and the assay works well with a variety of antiviral compounds.
Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , Enzyme-Linked Immunosorbent Assay/methods , Antigens, Viral/analysis , Cells, Cultured , Drug Evaluation, Preclinical/methods , Humans , Microbial Sensitivity Tests/methods , Viral Plaque AssayABSTRACT
The in vitro activity of the synthetic fluoroquinolone amifloxacin was compared with those of six other antibiotics: ampicillin, aztreonam, cefotaxime, cephalexin, cinoxacin, and gentamicin. Amifloxacin had the lowest 50% MIC of any of the antibiotics tested against aminoglycoside-resistant Pseudomonas aeruginosa, 4 micrograms/ml.