pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth

Abstract

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade.

Mouse Cytomegalovirus M34 Encodes a Non-essential, Nuclear, Early-Late Expressed Protein Required for Efficient Viral Replication

“…Chromatin immunoprecipitation (ChIP) experiments confirmed the binding of pUL34 to the predicted sites in the oriLyt and identified another binding site in the oriLyt harboring the similar but non-identical sequence motif AAACgCCGTc (Slayton et al, 2018). Site-directed mutagenesis of individual pUL34 binding sites within the oriLyt significantly attenuated HCMV replication (Slayton et al, 2018), suggesting a relevant role of pUL34-oriLyt associations. However, a mass spectrometry (MS)-based assessment of proteins associated with cell-free HCMV virions did not identify pUL34 as constituent of the HCMV particle (Varnum et al, 2004), implying that pUL34 may be stripped of the HCMV genome before or during packaging.…”

“…During HCMV infection, pUL34 acts as transcriptional repressor for viral genes such as US3 and US9. Additionally, pUL34 binds the oriLyt region (LaPierre and Biegalke, 2001;Liu and Biegalke, 2013) and enhances the efficiency of oriLyt-dependent DNA replication of plasmids (Slayton et al, 2018). Site-directed mutagenesis of the pUL34 binding sites in the oriLyt of HCMV reduced HCMV replication (Slayton et al, 2018).…”

“…The HCMV genome of strain AD169 contains 14 pUL34 consensus binding sites (consensus motif AAACACCGT[G/T]), three of which reside in the region of the origin of lytic replication (oriLyt) (Liu and Biegalke, 2013). Chromatin immunoprecipitation (ChIP) experiments confirmed the binding of pUL34 to the predicted sites in the oriLyt and identified another binding site in the oriLyt harboring the similar but non-identical sequence motif AAACgCCGTc (Slayton et al, 2018). Site-directed mutagenesis of individual pUL34 binding sites within the oriLyt significantly attenuated HCMV replication (Slayton et al, 2018), suggesting a relevant role of pUL34-oriLyt associations.…”

“…However, a mass spectrometry (MS)-based assessment of proteins associated with cell-free HCMV virions did not identify pUL34 as constituent of the HCMV particle (Varnum et al, 2004), implying that pUL34 may be stripped of the HCMV genome before or during packaging. Additionally, pUL34 physically interacts with pIE2, pUL44, and pUL84 (Slayton et al, 2018).…”

Functional Profile of Human Cytomegalovirus Genes and Their Associated Diseases: A Review

“…A wide range of viral genes are involved in viral replication. For instance, HCMV UL34 encodes a sequence-specific DNA binding protein (pUL34), which interacts with pUL84, pIE2, and pUL44 and contributes to the establishment of a nuclear environment necessary for viral gene expression and DNA replication (Rana and Biegalke, 2014;Slayton et al, 2018). pUL44 is an obligate nuclear-resident, non-structural viral protein for HCMV DNA replication (Neo et al, 2019).…”