Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome.
We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.
Kohler
C P
CP
Department of Microbiology and Immunology, Eastern Virginia Medical School, Norfolk 23501.
Kerry
J A
JA
Carter
M
M
Muzithras
V P
VP
Jones
T R
TR
Stenberg
R M
RM
D015139:Blotting, Southern; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005347:Fibroblasts; D015870:Gene Expression; D005819:Genetic Markers; D016679:Genome, Viral; D005966:Glucuronidase / Q000032:analysis / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008297:Male; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012867:Skin; D014763:Viral Matrix Proteins / Q000235:genetics
D004279:DNA, Viral; D005819:Genetic Markers; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; D015500:Chloramphenicol O-Acetyltransferase; D004259:DNA-Directed DNA Polymerase; D005966:Glucuronidase