Sumoylation of the Carboxy-Terminal of Human Cytomegalovirus DNA Polymerase Processivity Factor UL44 Attenuates Viral DNA Replication

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.

Site-specific SUMOylation of viral polymerase processivity factor: a way of localizingtoND10 subnuclear domains for restricted and self-controlled reproduction of herpesvirus

“…UL44 protein totally contains 31 lysines. Among them, using SUMOsp 2.0 prediction software [ 54 ], we previously found only one canonical SCM (SUMO Conjugation Motif; ψ 410 KxE) site at 410 lysine, which functions as the major SUMOylation site of UL44 to attenuate HCMV replication [ 35 ]. Sequence alignments showed that this canonical ψ 410 KxE site is single and strictly conserved among UL44 and other CMV homologues, lying just adjacent to the UL44 NLS motif at C-terminal end ( Figure 4a ).…”

“…As shown in Figure 4b , substitution of the major SUMOylated 410 lysine residue with arginine led to a great decline in UL44-SUMO1 level (lane 4 versus lane 2). However, PIAS3 lost the capability to enhance UL44 SUMOylation at other lysine residues outside the SCM site, even unable to recover the SUMOylation level of K410R mutant to that of WT (lane 5 versus lane 4 in Figure 4b ); this is distinct from UBC9, which could still partially recover the SUMOylation capability of UL44-K410A mutant [ 35 ]. In addition, HCMV infection assays also hinted the physiological correlation of UL44 SCM site with PIAS3.…”

“…Notably, we previously found the SUMOylation at K410 of UL44 could attenuate HCMV replication, and thus v-K410R, the mutated virus, owns a quicker viral DNA synthesis rate [ 35 ] (Fig S3, a). For this end, to exclude the possibility that UL44-wt and UL44-K410R distributions of nuclear foci and nuclear diffusion might, respectively, reflect viral pre-RC loci and the later viral DNA replication stage ( Figure 5b ; Fig S1), we also constructed U251-siUL54 host cells (Fig S2), in which no matter infected with wt-HCMV or v-K410R, the viral DNA synthesis was largely blocked by siUL54 (interfering in HCMV DNA polymerase) similarly (Fig S3, a).…”

“…293 T cells were co-transfected with pCMV-Myc-UL44 or pCMV-Myc-UL44-K410R, pcDNA-His-PIAS3 (wild-type or C334S mutant) and other SUMOylation-related plasmids (pRK-Flag-SUMO-1/-2/-3, pcDNA-HA-UBC9) as indicated using Lipofectamine 2000 (Invitrogen). After 48 h, the in vivo SUMO modifications of UL44 and its K410R mutant in transfected cells were analyzed by Western blot using anti-Myc and anti-Flag antibodies, as described in our previous work [ 35 ]. Meanwhile, to analyze the SUMOylation of UL44 under HCMV infection, U251 cells were first transfected with siControl or siPIAS3, and 24 h later infected with wt-HCMV or v-K410R at an MOI of 1.…”

“…Until recently, we found 410 lysine within a canonical SCM (ψ 410 KxE) site, located adjacent to the NLS motif of UL44, is the protein’s major SUMO modification site, which has no impacts on UL44 subcellular localization or protein stability but can attenuate the viral DNA replication upon its SUMOylation [ 35 ]. However, the selective mechanism by which SUMO is preferentially modified at K410 residue of UL44, as well as how this SCM-specific SUMOylation on UL44 can negatively regulate HCMV replication and reproduction, still remains unclear.…”