ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus - PubMed (original) (raw)

ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of Sindbis virus

Nadia V Giannakopoulos et al. J Virol. 2009 Feb.

Abstract

Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-alphabetaR(-/-)) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-alphabetaR(-/-) mice or prolong survival of ISG15(-/-) mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.

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Figures

FIG. 1.

FIG. 1.

Mutation of UbE1L-interacting residue arginine 153 in hISG15 affects protein conjugate formation. (A) Western blot analysis of six-His hISG15 (GG) and six-His hISG15 UbE1L-interacting mutant R153A following cotransfection with hUbE1L, UbcH8, and Herc5 into 293T cells. Asterisks indicate nonspecific bands detected in mock-transfected samples, and numbers (for panels A, C, D, and E) represent molecular mass markers (kDa). (B) Alignment of hISG15 and mISG15 sequences. Highlighted amino acids indicate ISG15 residues predicted to interact with UbE1L from hISG15 crystallization studies (33). R153A and R151A indicate hISG15 and mISG15 residues mutated in this study. (C and D) Anti-mISG15 Western blot analysis of 293T cells transfected with mISG15 LRLRGG (GG), conjugation-deficient mISG15 LRLRAA (AA), and UbE1L-interacting mutant mISG15 R151A (R151A) in the absence (C) or presence (D) of an E3 ligase, Herc5. (E) Representative anti-mISG15 Western blot analysis of IFN-β-treated ISG15−/− MEFs transfected with mISG15 LRLRGG (GG), mISG15 LRLRAA (AA), or mISG15 R151A (R151A) (top panel). The blot was reprobed for GFP (bottom panel). (F) Quantization of Western blot signals in panel E. Error bars represent the standard errors of the means from four experiments. One-sample t tests indicated that both mISG15 LRLRAA and mISG15 R151A (asterisks) were significantly different from mISG15 LRLRGG (mISG15 LRLRAA, P = 0.0015; 95% confidence interval, −0.264 to 4.294; mISG15 R151A, P = 0.0045; 95% confidence interval, 0.876 to 6.199). The graph is an arbitrary scale with mISG15 LRLRGG assigned a value of 10.

FIG. 2.

FIG. 2.

Mutation of UbE1L-interacting residue Arg151 attenuates mISG15's antiviral function during Sindbis virus infection. (A) Western blot analysis of BHK-21 cells mock infected (M) or infected with parental dsTE12Q (Q) or dsTE12Q containing mISG15 LRLRGG (GG), mISG15 LRLRAA (AA), or mISG15 R151A (R151A) at an MOI of 20. Parallel blots were probed with anti-ISG15 (top panel) or polyclonal anti-Sindbis virus (bottom panel). (B) Single-step growth curves of parental (dsTE12Q) or recombinant (mISG15 LRLRGG, mISG15 LRLRAA, or mISG15 R151A) Sindbis viruses in BHK-21 cells (MOI, 5). Data are represented as means ± standard errors of the means for three (dsTE12Q) or six (recombinant viruses) replicates. There are no significant differences between the medians of different viruses when analyzed by analysis of variance (P = 0.9993). (C) Survival of IFN-αβR−/− mice infected with 5 × 106 PFU of Sindbis viruses s.c. as indicated. The number of mice infected per group is indicated in parentheses, and data are compiled from a minimum of three experiments. Asterisks indicate curves statistically different from mISG15 LRLRGG (LRLRGG versus R151A, LRLRGG versus LRLRAA, and LRLRGG versus dsTE12Q, all P < 0.001).

FIG. 3.

FIG. 3.

ISG15 UbE1L-interacting mutant R151A is attenuated in its ability to decrease Sindbis virus replication. Viral titers of dsTE12Q, mISG15 LRLRGG, or mISG15 R151A in brains, spleen, liver, lung, and serum of IFN-αβR−/− mice infected for 3, 5, or 7 days. Data are pooled from two independent experiments with six mice per group. Error bars represent standard errors of the means, and the dashed line indicates the plaque assay limit of detection. Brackets denote statistically significant (P < 0.05) differences between indicated viruses.

FIG. 4.

FIG. 4.

mISG15 UbE1L-interacting mutants prolong survival during Sindbis virus infection. Survival of ISG15−/− pups infected with 1,000 PFU intracerebrally of virus expressing either mISG15 LRLRGG, mISG15 LRLRAA, or mISG15 R151A. Data are pooled from two (mISG15 R151A) or three (mISG15 LRLRGG and mISG15 LRLRAA) experiments, and the numbers of mice per group are indicated in parentheses. P values for comparisons between survival curves are as follows: mISG15 R151A versus mISG15 LRLRGG, P = 0.0002; mISG15 R151A versus mISG15 LRLRAA, P = 0.0365; mISG15 LRLRGG versus mISG15 LRLRAA, P < 0.0001.

FIG. 5.

FIG. 5.

UbE1L−/− mice display increased susceptibility to Sindbis virus infection. (A) Anti-mISG15 Western blot analysis of brains from BL6 or UbE1L−/− mice infected with 5 × 106 PFU of dsTE12Q s.c. on day 3 or 7 postinfection. Parallel blots were probed with anti-mISG15 (top panel) or anti-β-actin (bottom panel). DPI, day postinfection. (B) Survival of UbE1L−/− and BL6 mice following infection with 5 × 106 PFU of dsTE12Q s.c. P values of log-rank comparisons between UbE1L−/− and BL6 mouse survival curves are shown.

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