Chen Jun, Li Guanlie, He Haiqing, Li Xin, Niu Wenjing, Cao Di, Shen Ao
Key Laboratory of Molecular Target and Clinical Pharmacology, State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China.
Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, China.
Front Microbiol. 2021 Apr 21;12:652719. doi: 10.3389/fmicb.2021.652719. eCollection 2021.
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 His-tag pull-down and 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 and sumoylation assays, as well as in HCMV-infected cells. We further confirmed that lysine located within a ψKxE consensus motif on UL44 carboxy-terminal was the major sumoylation site of UL44. Interestingly, although lysine had no effects on subcellular localization or protein stability of UL44, the removal of lysine 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.
对基因组DNA合成进行可控调节是包括人类巨细胞病毒（HCMV）在内的所有疱疹病毒普遍保守的过程，并且在病毒发病机制（如持续性感染）中起关键作用。HCMV DNA聚合酶持续合成因子UL44在病毒DNA复制中起重要作用。为了更好地了解UL44的生物学特性，我们进行了酵母双杂交筛选，以寻找可与UL44相互作用的宿主蛋白。最常分离到的结果是SUMO缀合酶UBC9，一种参与SUMO化途径的蛋白。通过His标签下拉和共免疫沉淀试验证实了UBC9与UL44的相互作用。使用UL44的缺失突变体，我们确定了UL44的两个小区域，即第11 - 16位氨基酸和第260 - 269位氨基酸，这可能对与UBC9的相互作用至关重要。然后我们通过体外SUMO化试验以及在HCMV感染的细胞中证明UL44是SUMO化的靶标。我们进一步证实，位于UL44羧基末端ψKxE共有基序内的赖氨酸是UL44的主要SUMO化位点。有趣的是，尽管赖氨酸对UL44的亚细胞定位或蛋白质稳定性没有影响，但去除赖氨酸SUMO化位点增强了HCMV在转染 - 复制试验中的病毒DNA合成以及在感染细胞中的病毒子代产生，这表明SUMO化可通过靶向UL44来减弱HCMV复制。我们的结果表明SUMO化在调节UL44功能和病毒复制中起关键作用，并揭示了UL44羧基末端的关键作用，此前其功能鲜为人知。
