Open AccessThe Human Cytomegalovirus Gene UL79 Is Required for the Accumulation of Late Viral Transcripts
Author(s) -
Yi-Chieh Perng,
Qian Zhou,
Anthony R. Fehr,
Baoqin Xuan,
Dong Yü
Publication year - 2011
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.02344-10
Subject(s) - biology , viral replication , viral structural protein , human cytomegalovirus , virology , stop codon , open reading frame , gene , hemagglutinin (influenza) , mutation , mutant , virus , viral protein , viral entry , microbiology and biotechnology , genetics , peptide sequence
In this study, we adopted a conditional protein genetic approach to characterize the role of the human cytomegalovirus (HCMV) gene UL79 during virus infection. We constructed ADdd UL79, a recombinant HCMV in which the annotated UL79 open reading frame (ORF) was tagged with the destabilization domain of a highly unstable variant of the human FKBP12 protein (dd FKBP). Thedd FKBP domain targets the tagged protein for rapid proteasomal degradation, but the synthetic ligand Shield-1 can stabilizedd FKBP, allowing accumulation of the tagged protein. ADdd UL79 failed to replicate without Shield-1, but it grew at wild-type levels with Shield-1 or in human foreskin fibroblasts overexpressing hemagglutinin (HA)-tagged UL79 (HF-UL79HA cells), indicating an essential role of UL79 and the effectiveness of this approach. Without Shield-1, representative immediate-early and early viral proteins as well as viral DNA accumulated normally, but late transcripts and proteins were markedly reduced. UL79 was transcribed with early-late kinetics, which was also regulated via a positive-feedback loop. Using HF-UL79HA cells, we found that the UL79 protein localized to viral replication compartments during HCMV infection. Finally, we created a second UL79 mutant virus (ADin UL79stop ) in which the UL79 ORF was disrupted by a stop codon mutation and found that ADin UL79stop phenocopied ADdd UL79 under the destabilizing condition. Taking these results together, we conclude that UL79 acts after viral DNA replication to promote the accumulation of late viral transcripts. Importantly, the comparative analysis of ADdd UL79 and ADin UL79stop viruses provide additional proof for the power of the protein stability-based conditional approach to dissect the role of viral factors in HCMV biology.