Abstract
Controlled regulation of genomic DNA synthesis is a universally conserved process for all herpesviruses, including human cytomegalovirus (HCMV), and plays a key role in viral pathogenesis, such as persistent infections. HCMV DNA polymerase processivity factor UL44 plays an essential role in viral DNA replication. To better understand the biology of UL44, we performed a yeast two-hybrid screen for host proteins that could interact with UL44. The most frequently isolated result was the SUMO-conjugating enzyme UBC9, a protein involved in the sumoylation pathway. The UBC9-UL44 interaction was confirmed by in vitro His-tag pull-down and in vivo co-immunoprecipitation assays. Using deletion mutants of UL44, we mapped two small regions of UL44, aa 11–16, and 260–269, which might be critical for the interaction with UBC9. We then demonstrated that UL44 was a target for sumoylation by in vitro and in vivo sumoylation assays, as well as in HCMV-infected cells. We further confirmed that 410lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although 410lysine had no effects on subcellular localization or protein stability of UL44, the removal of 410lysine sumoylation site enhanced both viral DNA synthesis in transfection-replication assays and viral progeny production in infected cells for HCMV, suggesting sumoylation can attenuate HCMV replication through targeting UL44. Our results suggest that sumoylation plays a key role in regulating UL44 functions and viral replication, and reveal the crucial role of the carboxy-terminal of UL44, for which little function has been known before.
Highlights
Human cytomegalovirus (HCMV), a member of β-herpesviruses, is the leading viral cause of congenital abnormalities and intellectual disability in newborns, and it causes severe lifethreatening complications in immunocompromised individuals like AIDS patients and transplant recipients (Mocarski et al, 2007)
UL44 carboxy-terminal was found to be indispensable for virus replication and for the formation of DNA replication compartments in infected cells (Kim and Ahn, 2010; Silva et al, 2010), even when this segment was replaced with another nuclear localization signal (NLS) that ensured nuclear localization (Silva et al, 2010)
To identify cellular proteins interacting with human cytomegalovirus (HCMV) UL44, we used advanced GAL4-based yeast two-hybrid (YTH) system with yeast strain AH109 and three reporters (ADE2, HIS3, and lacZ)
Summary
Human cytomegalovirus (HCMV), a member of β-herpesviruses, is the leading viral cause of congenital abnormalities and intellectual disability in newborns, and it causes severe lifethreatening complications in immunocompromised individuals like AIDS patients and transplant recipients (Mocarski et al, 2007). UL44 carboxy-terminal was found to be indispensable for virus replication and for the formation of DNA replication compartments in infected cells (Kim and Ahn, 2010; Silva et al, 2010), even when this segment was replaced with another NLS that ensured nuclear localization (Silva et al, 2010) This clearly argues for a crucial role for the carboxy-terminal of UL44 in HCMV viral DNA synthesis and viral replication, beyond its role in nuclear localization. It was proposed that the carboxy-terminal segment might interact with host or viral proteins involved in DNA replication (Kim and Ahn, 2010), the exact role of the carboxy-terminal of UL44 remains to be elucidated far
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