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. 2017 Feb 20;14(1):34.
doi: 10.1186/s12985-016-0673-5.

Interaction between HCMV pUL83 and human AIM2 disrupts the activation of the AIM2 inflammasome

Yuan Huang  1 , Di Ma  1 , Heyu Huang  1 , Yuanyuan Lu  1 , Yi Liao  1 , Lingling Liu  1 , Xinglou Liu  1 , Feng Fang  2   3
Affiliations

Interaction between HCMV pUL83 and human AIM2 disrupts the activation of the AIM2 inflammasome

Yuan Huang et al. Virol J. .

Abstract

Background: AIM2, a cytosolic DNA sensor, plays an important role during infection caused by pathogens with double-stranded DNA; however, its role in human cytomegalovirus (HCMV) infection remains unclear. Previously, we showed an increase in AIM2 protein levels during the early stage of HCMV infection and a decrease 24 h post infection. Because HCMV has developed a variety of strategies to evade host immunity, we speculated that this decline might be attributed to a viral immune escape mechanism. The tegument protein pUL83 is an important immune evasion protein and several studies have reported that pUL83 binds to specific cellular proteins, such as AIM2-like receptor IFI16, to affect their functions. To determine whether pUL83 contributes to the variation in AIM2 levels during HCMV infection, we investigated the pUL83/AIM2 interaction and its impact on the AIM2 inflammasome activation.

Methods: We constructed plasmids expressing recombinant pUL83 and AIM2 proteins for two-hybrid and chemiluminescence assays. Using co-immunoprecipitation and immunofluorescent co-localization, we confirmed the interaction of pUL83/AIM2 in THP-1-derived macrophages infected with HCMV AD169 strain. Furthermore, by investigating the expression and cleavage of inflammasome-associated proteins in recombinant HEK293T cells expressing AIM2, apoptosis-associated speck-like protein (ASC), pro-caspase-1 and pro-IL-1β, we evaluated the effect of pUL83 on the AIM2 inflammasome.

Results: An interaction between pUL83 and AIM2 was detected in macrophages infected with HCMV as well as in transfected HEK293T cells. Moreover, transfection of the pUL83 expression vector into recombinant HEK293T cells stimulated by poly(dA:dT) resulted in reduced expression and activation of AIM2 inflammasome-associated proteins, compared with the absence of pUL83.

Conclusions: Our data indicate that pUL83 interacts with AIM2 in the cytoplasm during the early stages of HCMV infection. The pUL83/AIM2 interaction deregulates the activation of AIM2 inflammasome. These findings reveal a new strategy of immune evasion developed by HCMV, which may facilitate latent infection.

Keywords: AIM2 inflammasome; HCMV; Immune evasion; pUL83.

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Figures

Fig. 1
Fig. 1
Construction and expression of recombinant UL83 and AIM2 proteins. a UL83 ORF (1686 bp) was cloned into the MCS of the pM vector for the expression of a fusion of a bait protein (pUL83 herein) with Gal4 DNA BD (147 aa). b AIM2 ORF (1024 bp) was inserted into pVP16 vector to express recombinant AIM2 (the prey protein) fused to VP16 AD (45 aa). c Plasmids digested by EcoRI and SalI or EcoRI and BamHI and PCR products were analyzed by agarose gel electrophoresis. The sizes of the plasmid digestion and PCR products were as anticipated. d Verified recombinants were used to transfect HEK293T cells for 72 h. The expression of target proteins was assessed by SDS-PAGE with specific antibodies against pUL83 and AIM2. The expected recombinant protein sizes were 81 kDa (BD-pUL83) and 44 kDa (AD-AIM2)
Fig. 2
Fig. 2
Detection of the interaction between pUL83 and AIM2. a Schematic diagram of a two-hybrid experiment, adapted from the Matchmaker Mammalian Assay Kit. b Plasmids encoding recombinant pUL83 and AIM2 proteins were used together with pG5SEAP to co-transfect HEK293T cells for 72 h. Supernatants were then collected and SEAP levels were detected by chemiluminescence at 405 nm. The experiment was repeated three times. Statistical data were analyzed using the t-test. c Plasmids encoding recombinant pUL83 and AIM2 were used to transfect HEK293T cells for 72 h. Cells were harvested and lysed with protein lysis buffer, and whole cell lysates were immunoblotted using specific antibodies against pUL83 and AIM2, or immunoprecipitated with the anti-AIM2 antibody and then detected using the anti-pUL83 antibody. IgG was used as a negative control. Data from one representative experiment out of three are presented as the mean ± SD. * P < 0.01. WCL: whole cell lysates
Fig. 3
Fig. 3
Detection of the pUL83/AIM2 interaction in HCMV-infected cells. a THP-1 cells were stimulated with PMA (100 ng/mL) to induce cellular differentiation. They were then mock-infected or infected with the HCMV AD169 strain for 6 h, 12 h, or 24 h, or transfected with poly(dA:dT). The cells were harvested and lysed and whole-cell lysates were immunoblotted using specific antibodies against pUL83 and AIM2, or immunoprecipitated with the anti-AIM2 antibody and then detected with anti-pUL83 and anti-AIM2 antibodies. b–d The infected cells were washed and fixed at the indicated time points. Specific antibodies against pUL83 and AIM2 were added and then conjugated with fluorescently tagged secondary antibodies. Cell nuclei were stained with DAPI. P: poly(dA:dT). WCL: whole cell lysates. Red, AIM2; green, pUL83; DAPI (blue), nuclei
Fig. 4
Fig. 4
The effect of the pUL83/AIM2 complex on the AIM2 inflammasome. ASC, pro-caspase-1, and pro-IL-1β ORFs were inserted into pDsRed2-N1 expression vectors. The recombinant vectors with or without the pVP-AIM2 vector were used to co-transfect HEK293T cells transiently for 72 h. The resultant cells were named rHEK293T or rHEK293T (AIM2-). Protein expression of these cells (a and b, line 2 and 7) as well as that of wild HEK293T cells (a and b, line 1) was determined by immunoblotting. Poly(dA:dT) was used to transfect rHEK293T cells for 6 h and 24 h or transfect rHEK293T (AIM2-) cells for 6 h to activate the AIM2 inflammasome, and the expression and activation of inflammasome proteins were then determined (a and b, lines 3, 4 and 8). The UL83 expression vector was used to transfect rHEK293T cells, which were then stimulated with poly(dA:dT); the inflammasome proteins were then detected (a and b, lines 5, 6 and 9). P: poly(dA:dT). p10: the activated form of caspase-1
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