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. 2009 Dec;83(24):12881-94.
doi: 10.1128/JVI.01525-09. Epub 2009 Oct 7.

Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection

Anja Berndt  1 , Heike Hofmann-Winkler, Nina Tavalai, Gabriele Hahn, Thomas Stamminger
Affiliations

Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection

Anja Berndt et al. J Virol. 2009 Dec.

Abstract

The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.

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Figures

FIG. 1.
FIG. 1.
Identification of a putative SIM (boldfaced) within the IE2p86 sequence by bioinformatics approaches and yeast two-hybrid analyses. (A) Comparison of the putative SIM of IE2p86 to SIMs of hDaxx and human PML (hPML). (B) Site-directed mutagenesis was used to introduce the amino acid changes I200A and V201A into the IE2p86 sequence in order to disrupt the putative IE2p86 SIM. The disruption of both IE2p86 SUMOylation sites was described previously (17). The IE2p86 sequence with the mutated SIM was termed IE2M2 and the IE2p86 sequence with the mutated SIM and SUMOylation sites was termed IE2ΔSM2. Mutated amino acids are shown in boldface. (C) Yeast two-hybrid analyses with various IE2p86 point and deletion mutants fused to the GAL4 DNA binding domain (BD-IE2p86), together with SUMO fused to the GAL4 activation domain (AD-SUMO1), were performed to further determine the SUMO-IE2p86 interaction. The expression of the reporter genes ADE2 (growth on −AWL) and lacZ (β-Gal-Assay) was observed under high-stringency growth conditions. The Roman numerals refer to the IE2p86 mutants that were investigated for interaction with SUMO1.
FIG. 2.
FIG. 2.
Coimmunoprecipitation of IE2p86 and SUMO1 from transiently transfected HEK-293T cells. HEK-293T cells were cotransfected with vectors expressing Flag-SUMO1 and wild-type or mutant IE2p86 as indicated. (A) Coimmunoprecipitations were performed using an anti-IE2p86 antibody, and the precipitates were analyzed by Western blotting using an anti-Flag MAb. The squares indicate the positions of SUMOylated isoforms of IE2p86. (B) The protein input of this experiment was analyzed by Western blotting using either anti-FLAG antibody (top) or an anti-IE2p86 rabbit antiserum (middle). Beta-actin was detected as a loading control (bottom).
FIG. 3.
FIG. 3.
Construction and verification of recombinant HCMVs expressing SUMOylation-negative IE2p86. (A) Schematic diagram illustrating the generation of recombinant viruses by homologous recombination in E. coli. First, a selection marker, either the kanamycin resistance or the galK gene, was inserted into exon 5 of the ie1/2 region of BAC pHB5 or FIX-BAC, respectively. Subsequently, the selection marker was replaced by either a wild-type or a mutated IE2p86 sequence. The resulting BACs, AD169 based (B and C) or FIX-BAC based (D), were digested with either EcoRI (B) or BamHI (C and D) and analyzed by agarose gel electrophoresis. (B) When digested with EcoRI, after successful reintroduction of either the wild-type (lanes 2 and 3) or mutant (lanes 4 and 5) exon 5 IE2p86 region into ΔkΔex5, the 8-kb band (indicated by a triangle in lane1) was shifted to approximately 10 kb (indicated by a circle in lane 2). (C) When digested with BamHI, an additional 5-kb band appeared (lanes 2 to 6, indicated by a circle). (D) Introduction of the galK sequence in FIX-BAC resulted in the loss of an approximately 5-kb band (indicated by a square in lane 1) and the reappearance of this band when either the wild-type (lanes 5 and 6) or mutant (lanes 3 and 4) IE2p86 sequence was reinserted into the BAC.
FIG. 4.
FIG. 4.
Growth kinetics of recombinant AD169-derived viruses. In order to determine the replication capacities of the recombinant viruses AD169, Ad169rev, and AD169ex-ΔS, a multistep growth curve analysis was performed. HFFs were infected with equal IE units (MOI, 0.01) of wild-type (AD169), revertant (AD169rev), or mutant (AD169ex5-ΔS) recombinant virus. The viral supernatants were harvested at the indicated time points (d, day), followed by the determination of viral titers. Each infection was performed in triplicate, and the standard deviations are depicted by error bars.
FIG. 5.
FIG. 5.
Impaired initiation of IE gene expression in AD169- and FIX-BAC-derived viruses with mutated SUMOylation acceptor sites. (A and B) HFFs were infected with equivalent IE units of AD169- and FIX-BAC-derived recombinant wild-type and mutant viruses. Total intracellular DNA was isolated 14 h p.i., followed by the determination of viral genomic equivalents by HCMV-specific TaqMan PCR. (C) The numbers of viral plaques following infection with equal genomic equivalents of FIX-BAC, FIX-BACrev, and FIX-BACex5-ΔS. HFFs were infected with viral genomic equivalents of each recombinant virus (corresponding to 200 PFU of FIX-BAC virus), followed by the addition of overlay medium. Plaques were counted at 5, 7, 9, and 14 days (d) p.i. All experiments were performed in triplicate, and standard deviations are indicated by error bars.
FIG. 6.
FIG. 6.
Comparison of the growth of AD169ex5-ΔS and AD169ex5-M2 and the kinetics and levels of intracellular viral DNA of AD169 and AD169ex5-ΔSM2. (A) To compare the replication capacities of the wild-type and mutant viruses AD169ex5-ΔS and AD169ex5-ΔSM2, a multistep growth curve analysis was performed. HFFs were infected in parallel with AD169, AD169ex-ΔS, and AD169ex-ΔSM2 (MOI, 0.01). The viral supernatants were collected during a period of 12 days (d) p.i., and the viral titers were obtained by determination of IE1p72-positive cells. (B) The replication capacities of AD169, AD169ex5-ΔS, and AD169ex5-M2 were compared as described for panel A, with the exception that viral supernatants were collected during a period of 14 days p.i. (C) Determination of intracellular viral DNA following infection with equal IE units of AD169, AD169ex-ΔS, and AD169ex-ΔSM2 by HCMV-specific TaqMan PCR. HFFs were infected with 30,000 IE units of the respective recombinant virus, and total intracellular DNA was harvested and analyzed 10 h p.i. as described in Materials and Methods. For all experiments, standard deviations are depicted by error bars.
FIG. 7.
FIG. 7.
Accumulation of viral-progeny DNA after infection with AD169- and FIX-BAC-derived viruses. The accumulation of viral-progeny DNA in cells infected with AD169-derived (A and C) and FIX-BAC-derived (B) recombinant viruses was measured by TaqMan-PCR. HFFs were infected in parallel with equal genomic equivalents of wild-type and mutant viruses, and total intracellular DNA was harvested over a period of 120 h p.i. as indicated. All experiments were performed in triplicate, and standard deviations are depicted by error bars.
FIG. 8.
FIG. 8.
Subnuclear localization of IE2p86 in HFFs after infection with wild-type, revertant, and mutant recombinant viruses. HFFs were infected at an MOI of either 1.5 (A and B) or 1 (C) with AD169, AD169rev, AD169ex5-ΔS, AD169ex5-M2, or AD169ex5-ΔSM2. The cells were fixed with paraformaldehyde at either 240 min (A and B) or 24, 72, and 96 h (C) p.i. Nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole), and the cellular nuclear domain 10 component PML was detected with monoclonal anti-PML antibody PG-M3. IE2p86 was detected by the polyclonal antiserum anti-pHM178. The inset images show regions of the cell at a higher magnification. (B) In order to obtain mean values for the number of IE2 dots per cell, 50 infected cells were randomly chosen, and the dots were counted. Standard deviations are depicted by error bars. (D) HFFs were infected at an MOI of 1 with AD169, AD169rev, AD169ex5-ΔS, or AD169ex5-ΔSM2, respectively. Forty-eight hours after infection, the cells were fixed with paraformaldehyde and stained with MAb-UL44 to detect viral replication compartments. Thereafter, 50 cells were inspected with respect to the presence of either prereplication foci or replication compartments. The graph indicates the relative distribution of prereplication foci and replication compartments in the infected cell population.
FIG. 9.
FIG. 9.
Analysis of viral-protein expression in AD169-, AD169ex5-M2-, AD169ex5-ΔS-, and AD169ex5-ΔSM2-infected HFFs. (A and B) HFFs were infected in parallel with wild-type or recombinant viruses (MOI, 0.3 for panel A and 0.1 for panel B) to investigate the viral-protein expression kinetics by Western blot analysis. At 24, 48, 72, and 96 h p.i., cell lysates were prepared, subjected to SDS-polyacrylamide gel electrophoresis, and subsequently transferred onto a nitrocellulose membrane. IE (IE2p86 and IE1p72), early (UL44), early-late (UL69 and pp71), and true late (MCP and pp28) protein expression levels were analyzed by Western blotting as described in Materials and Methods. The cellular protein beta-actin served as a control for equal protein loading. (A) Protein expression levels of AD169 and AD169ex5-M2 were compared. (B) Protein expression levels of AD169, AD169ex5-ΔS, and AD169ex5-ΔSM2 were compared.
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