Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression.
The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections. We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions. Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359. The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles. The IE2 86 Delta 356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC. This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant. Both exhibit detectable upregulation of major immediate-early transcripts at early times. The IE2 86 Delta 427-435 and IE2 86 Delta 505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter. These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection. We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression.
White
Elizabeth A
EA
Molecular Biology Section and Center for Molecular Genetics, University of California-San Diego, La Jolla, California 92093-0366, USA.
Clark
Charles L
CL
Sanchez
Veronica
V
Spector
Deborah H
DH
D003587:Cytomegalovirus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000821:virology; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D015534:Trans-Activators; D014162:Transfection; D014779:Virus Replication
C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D015534:Trans-Activators