[Show abstract][Hide abstract] ABSTRACT: The promoter controlling the expression of the transcript encoding the major herpes simplex virus type 1 capsid protein (VP5, UL19) extends only 60 bases or so from a functional Sp1 site at --48 to include a cis-acting element 3' of the transcript start site. In the present communication, we report the generation of recombinant viruses bearing mutations between --6 and + 8 relative to the cap site in the VP5 promoter controlling expression of a reporter gene. Analysis of the effects of these mutations upon reporter gene expression along with the results of protein binding assays demonstrates that this cap transcription element functionally interacts with a cellular protein of a normal size of 40 kDa. Thus, like the strict late UL38 promoter characterized earlier, the late VP5 promoter has the essential properties of a cellular promoter.
Journal of Virology 04/1996; 70(3):1898-904. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 10/1994; 68(9):5738-47. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 04/1994; 68(3):1972-6. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 10/1993; 67(9):5109-16. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: transcript (LAT)isbounded on its5'endbya putative promoter region whichcontains sequenceelements similar to thosecharacterized fortheherpessimplex virus(HSV)LAT promoter. Usingthebacterial ,-galactosidase gene as a reporter, we haveassayed PRV LAT promoter activity inthegenomic environment inrecombinant HSVs.ThePRV LATpromoter-,-galactosidase reporter gene was recombined intotheterminal andinternal longrepeatregions (RLregions), replacing thenormalHSV LATpromoter, thecapsite, andthefirst 60bases oftheprimarytranscript. When recombined intotheRIregion, appreciable reporter geneexpression was observed following infection oftwocelllines ofneuronal origin; little or no activity was seen withthese recombinants following infection ofrabbit skinor mouse embryofibroblasts. No significant expression was seenwhenthepromoter was recombined intothegClocusinthelonguniqueregion inany ofthecelltypes utilized. Suchresults suggest thatthePRVlatency promoter contains neuronal cell-specific elements andthat theHSV RLregion provides an appropriate genomicenvironment forthemanifestation ofthatspecificity.