Sequences related to SUMO interaction motifs in herpes simplex virus 1 protein ICP0 act cooperatively to stimulate virus infection

Abstract

Herpes simplex virus type 1 immediate-early protein ICP0 is an E3 ubiquitin ligase of the RING finger class that degrades several cellular proteins during infection. This activity is essential for its functions in stimulating efficient lytic infection and productive reactivation from latency. ICP0 targets a number of proteins that are modified by the small ubiquitin-like SUMO family of proteins, and it includes a number of short sequences that are related to SUMO interaction motifs (SIMs). Therefore, ICP0 has characteristics that are related to those of cellular SUMO-targeted ubiquitin ligase enzymes. Here, we analyze the impact of mutation of a number of SIM-like sequences (SLSs) within ICP0 on HSV-1 replication and gene expression and their requirement for ICP0-mediated degradation of both sumoylated and unmodified promyelocytic leukemia (PML) and other sumoylated cellular proteins. One SLS in the central portion of the ICP0 sequence (SLS4) was found to be absolutely required for targeting cellular sumoylated species in general and sumoylated forms of PML other than those of PML isoform I. Mutation of a group of SLSs in the C-terminal quarter of ICP0 also reduced ICP0-mediated degradation of sumoylated PML in a cooperative manner. Although mutation of individual SLSs caused only modest decreases in viral replication, combined mutation of SLS4 with SLS sequences in the C-terminal quarter of the protein reduced plaque formation efficiency by up to two orders of magnitude. These results provide further evidence that the biological activities of ICP0 are connected with host cell sumoylation events.

Importance: Herpes simplex virus type 1 protein ICP0 plays important roles in regulating the initial stages of lytic infection and productive reactivation from latency. ICP0 mediates its effects through inducing the degradation of cellular proteins that have repressive effects on viral gene expression. An increasing number of cellular proteins are known to be sensitive to ICP0-mediated degradation; therefore, it is important to understand how ICP0 selects its substrates for degradation. This study identifies sequence motifs within ICP0 that are involved in targeting cellular proteins that are modified by the SUMO family of ubiquitin-like proteins and describes how mutation of combinations of these motifs causes a 100-fold defect in viral infectivity.

FIG 1

Locations of the mSLS mutations and their effects on viral gene expression. (A) A map of the coding sequence of ICP0, indicating the minimal RING domain from the first to last zinc-coordinating cysteine residues (residues 116 to 156); SIM-like sequence SLS4 (360 to 366); nuclear localization signal (nls; 501 to 510); core of the USP7 binding region (614 to 630); SIM-like sequences SLS5 (651 to 655), SLS6 (666 to 669), and SLS7 (681 to 684); and two point mutations adjacent to SLS6 previously defined by the mutation R8507 (670 and 672). The shaded area (633 to 720) indicates the region implicated in efficient localization of ICP0 to ND10 (PML NBs). (B and C) Comparison of the viral gene expression efficiencies of the mSLS viruses. HepaRG cells were infected at an MOI of 1 with the indicated viruses. Samples taken at the indicated time points were analyzed by Western blotting for ICP0, ICP4, UL42, and VP5 as representatives of the IE, early, and late classes of viral proteins. Actin provides the loading control. M, molecular size ladder.

FIG 2

Immunofluorescence analysis of wt HSV-1 and mutant viruses mSLS4, mSLS5/7, and mSLS4/5/7. HFs on coverslips were infected at an MOI of 2, and then samples were analyzed at 2 h, 4 h, and 6 h after infection by immunofluorescence staining for ICP0 (green) and PML (red). Separated channels are shown in greyscale, with the color merged image to the right. The uppermost row shows PML and ICP0 staining in uninfected control cells.

FIG 3

Immunofluorescence analysis of wt HSV-1 and mutant viruses R8507, mSLS6, and mSLS5/6/7. HFs on coverslips were infected at an MOI of 2, and then samples were analyzed at 2 h, 4 h, and 6 h after infection by immunofluorescence staining for ICP0 (green) and PML (red). Separated channels are shown in greyscale, with the color merged image to the right.

FIG 4

Degradation rates of endogenous PML by wt and mSLS mutant viruses. HepaRG cells were infected with the indicated viruses at an MOI of 2, and then samples harvested at 2 h, 4 h, and 6 h after infection were analyzed by Western blotting for endogenous PML (MAb 5E10), ICP0, and actin.

FIG 5

Specificity of targeting of PML.I, PML.II, and their SUMO-conjugated forms by wt and mSLS mutant viruses. (A to G) HepaRG cells expressing EYFP-PML.I (left) or EYFP-PML.II (right) at close to endogenous levels were infected with the indicated viruses at an MOI of 5, and then samples harvested at 2 h, 4 h, 6 h, and 8 h were analyzed by Western blotting for EYFP fusion proteins, ICP0, and actin. (H) Comparison of rates of degradation of EYFP-PML.I by wt and mSLS4/5/7 mutant viruses at an MOI of 2.

FIG 6

Degradation rates of high-molecular-weight SUMO2/3-conjugated proteins by wt and mSLS mutant viruses. HepaRG cells were infected with the indicated viruses at an MOI of 2, and then samples harvested at 2 h, 4 h, and 6 h after infection were analyzed by Western blotting for SUMO2/3, ICP0, and actin. (C) Infection of HFs by wt and mutant mSLS4 viruses at an MOI of 5, illustrating the substantial increase in high-molecular-weight SUMO conjugates that can occur at later times of mSLS4 infection.

FIG 7

Inhibition of recruitment of PML to viral genome foci by wt and mSLS mutant viruses. HFs on coverslips were infected at a low MOI, depending on the intrinsic plaque-forming efficiencies of the viruses, so that small virus plaques had been produced 24 h later. The cells were analyzed by immunofluorescence staining for PML (red) and ICP4 (green). Separated channels are shown in greyscale, with the color merged image to the right.

FIG 8

Inhibition of recruitment of SUMO2/3-conjugated proteins to viral genome foci by wt and mSLS mutant viruses. HFs on coverslips were infected at a low MOI, depending on the intrinsic plaque-forming efficiencies of the viruses, so that small virus plaques had been produced 24 h later. The cells were analyzed by immunofluorescence staining for SUMO2/3 (red) and ICP4 (green). Separated channels are shown in greyscale, with the color merged image to the right.

FIG 9

Influence of mutations in the C-terminal quarter of ICP0 on the interaction between ICP0 and SUMO1 in vitro. The C-terminal quarter of ICP0 (residues 594 to 775) and derivatives, including the indicated mutations, were expressed as GST fusion proteins in bacteria and then purified using glutathione beads. Samples of the beads were mixed with bacterial extracts containing His-tagged SUMO1, and the relative levels of bound SUMO1 were detected by Western blotting of purified washed beads (lower). The upper panel shows a Coomassie stain of the bead preparations, confirming equal loading. GST-Ubc9 and GST beads were used as positive and negative controls.

FIG 10

Comparison of the regions apart from the RING finger that are most highly conserved in the ICP0 proteins of HSV-1, HSV-2, and HVB. (A) A plot of sequence relatedness between the three proteins as compiled in Align-X (VectorNTI; Invitrogen). (B to E) Alignments and residue coordinates of the SLS4 (B), USP7 binding (C), SLS5/SLS6/SLS7/R8507 (HSV-1 ICP0 coordinates only) (D), and C-terminal regions (E) are shown, with completely conserved residues indicated by asterisks. Apart from the RING finger and its immediate downstream region (which has 66% identity between the three proteins over the 119-residue stretch corresponding to HSV-1 ICP0 coordinates 116 to 224; data not shown), there is only limited precise conservation in other regions of the proteins.