Abstract
UL34 is one of the ~50 genes of human cytomegalovirus (HCMV) required for replication in cell culture in human fibroblasts. UL34 encodes highly related early (UL34a) and late (UL34b) proteins that are virtually identical, with the early protein containing an additional 21 amino terminal amino acids. The UL34 proteins are sequence-specific DNA‑binding proteins that localize to the nucleus. The HCMV genome contains 14 to 15 UL34 binding sites; two of the UL34 binding sites contribute to transcriptional regulation of two other viral genes, US3 and US9. The roles of the remaining binding sites and the requirement for both UL34 proteins during viral infection remain unknown. We examined the contributions of the early and late UL34 proteins to viral replication by generating HCMV-containing bacterial artificial chromosomes with the initiation codon for the early or the late protein mutated. Neither virus was able to replicate, demonstrating that UL34 expression is required throughout the viral replication cycle. A marked decrease in viral gene expression for each of the mutants suggests that UL34 proteins may contribute generally to transcriptional regulation. Intracellular localization studies demonstrated that UL34 colocalizes with the major immediate early protein, IE2, and the viral DNA polymerase processivity factor, UL44, to viral DNA replication centers. In conclusion, sustained UL34 protein expression is required for viral replication. The sequence-specific DNA binding ability of UL34 proteins, their localization to viral DNA replication centers and their general effects on viral gene expressions suggests that UL34 proteins contribute to the establishment of a nuclear environment necessary for viral gene expression and DNA replication.
Highlights
Human cytomegalovirus (HCMV) is predominantly an opportunistic pathogen, causing clinically significant disease in people who have inadequate immune responses, including neonates, transplant recipients, and people with uncontrolled HIV infections [1]
HCMV-bacterial artificial chromosome (BAC) that either entirely lacked UL34 ( UL34), contained UL34 with a mutation in the ATG initiating translation of the early protein [ATG mutated to ATC
To examine the defect in viral replication associated with the absence of UL34 proteins, semi-quantitative RT-PCR reactions were performed on RNA samples extracted following the electroporation of the UL34-HCMV BACs into human fibroblasts
Summary
Human cytomegalovirus (HCMV) is predominantly an opportunistic pathogen, causing clinically significant disease in people who have inadequate immune responses, including neonates, transplant recipients, and people with uncontrolled HIV infections [1]. Despite the large genome size, only ~50 genes are required for HCMV replication in cell culture, suggesting that the remaining 70% of the genes contribute to replication and latency in the human host [3,4]. Two highly related proteins are encoded by the early and late transcripts, with the late protein (UL34b) identical to the early protein (UL34a) except for the absence of 21 amino terminal amino acids. Both UL34 proteins localize to the nucleus and are sequence-specific DNA-binding proteins that act as transcriptional repressors; the interaction of UL34 proteins with the UL34 binding sites in the US3 and US9 genes down-regulates their expression [6,7]. The experiments described here were undertaken to identify the contributions of each of the UL34 proteins to viral replication and to examine the intracellular localization pattern of UL34 proteins during infection
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