PREreview of A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: 

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection  

Jessie Kulsuptrakul, Elizabeth A. Turcotte, Michael Emerman* and Patrick S. Mitchell* doi: https://doi.org/10.1101/2022.10.04.510817

We will adhere to the Universal Principled (UP) Review guidelines proposed in: 

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029 

SUMMARY: CARD8 is thought to sense infection stress and stimulate inflammasome formation, but precise mechanisms remain to be elucidated. HIV-1 protease (HIV-1^PR) cleaves CARD8, resulting in proteasome-dependent degradation of the amino-terminal CARD8 fragment and liberation of the carboxy-terminal fragment that activates inflammasomes. Kulsuptrakul and colleagues build upon this intriguing finding to investigate evolutionary relationships between primate lentiviruses and CARD8 homologs from their host primates. They discovered that both HIV-1 and SIV^cpzproteases cleave human CARD8. By contrast, chimpanzee CARD8 was not cleaved by primate lentivirus proteases. Thus, even prior to the HIV-1 pandemic, SIV^cpz proteases could cleave and activate human CARD8. The authors provided evidence for CARD8-dependent inflammasome activation in an in vitro HIV-1 infection model. Complementation of CARD8 KO cells with WT human CARD8 restored inflammasome activation, whereas complementation with chimpanzee CARD8 or human CARD8 mutant constructs with substitutions that destroy the protease cleavage site did not. Together, these findings support a role for human CARD8 in activating the inflammasome upon HIV infection and suggest that, during spillover events, SIVcpz was well equipped to cleave human CARD8 and drive pathogenesis.

OVERALL ASSESSMENT: This study convincingly demonstrates that human CARD8 is susceptible to cleavage and activation by diverse primate lentivirus protease enzymes, whereas chimpanzee CARD8 is not. Overall, the data is quite convincing and clearly organized and rendered for readers, and positions lentivirus proteases and host CARD8 sensors as new players in the ‘genomes-in-conflict’ chess match. These conclusions could be strengthened by additional evidence for CARD8-mediated inflammasome activation.  

STRENGTHS: The manuscript is well-written. The rationale provided for the investigation of CARD8 in the context of HIV infection is sound. The data presented is well-organized, clear and well-controlled. The primary strength is the identification and careful mapping of viral protease-mediated cleavage and activation of human CARD8, whereas the closely related chimpanzee CARD8 resists cleavage and activation.  

WEAKNESSES: While the manuscript was well written, the introduction was quite brief. We suggest that the authors should provide some additional information about CARD8 and inflammasomes. Does CARD8 have additional roles in the cell beyond inflammasome activation? How does CARD8 fit into the context of a diverse array of inflammasome triggers? This kind of information would really help set the stage properly. The data in Figs. 3/4 could be strengthened by additional assays to measure inflammasome activation beyond IL-1β secretion and PI staining. Options include an IL-18 ELISA, LDH release assay, HMGB1 release assay, or caspase-1 cleavage assay (western blot). The provision of a second assay to corroborate inflammasome activation is consistent with field-specific standards.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1.   Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.5

·     Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.

·       Fig. 1B – the cartoon introduces a 2^nd PR cleavage site on CARD8 (site 88/89) that is not investigated further. Is this site relevant to this study? Do the amino acids at this site vary between humans and Old-world monkeys? The reader would benefit from some explanation about this protease cleavage site. 

·       Fig. 1C – We wondered whether treatment with a proteasome inhibitor could prevent degradation of CARD8 protein fragments and help us better understand cleavage patterns on western blots. Moreover, could the unknown band at ~40 kDa be further characterized, and does it relate to the N-terminal cleavage product?

·       Figs 1D/2B - The anti-vinculin loading control western blots for Figure 1D and Figure 2B are identical.

·       Fig. 3 – As mentioned above, abstract-level conclusions about inflammasome activation require better support. The data in Fig. 3 could be strengthened by additional assays to measure inflammasome activation beyond IL-1β secretion and PI staining. Options include an IL-18 ELISA, LDH release assay, HMGB1 release assay, or caspase-1 cleavage assay (western blot). The provision of a second assay to corroborate inflammasome activation is consistent with field-specific standards.

·       Figs. 4B/4C – “Similar to our observations with VbP, we found that IL-1β secretion and cell death were significantly reduced in Pam3CSK4-primed, HIV-1LAI or HIV-1LAI-VSVG infected CARD8 KO versus WT THP-1 cells (Figure 4C). These results indicate that HIV-1-induced inflammasome activation in THP-1 cells is dependent on CARD8.” - This statement requires greater support demonstrating CARD8-dependent inflammasome activation. Inflammasome activation was still evident in the CARD8 KO cells. This analysis could benefit from corroboration with a second assay, as mentioned above. We wondered whether it would be feasible to employ a reconstructed inflammasome model, as reported in this recent paper:

o    Qiyao Chai et al. A bacterial phospholipid phosphatase inhibits host pyroptosis by hijacking ubiquitin. Science 378, eabq0132(2022). DOI:10.1126/science.abq0132

·       Fig. 4D - We were once again curious about the identity of the unknown ~40 kDa band, which is clearly present in the CARD8 KO lysates. Perhaps this could be addressed in the Discussion.                                                                     

Are specific analyses performed using methods that are consistent with answering the specific question?  

·     Is there appropriate technical expertise in the collection and analysis of data presented? 

·   Yes

·     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?  

·   Yes

·     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.  

·   The provision of a second assay to corroborate inflammasome activation is consistent with field-specific standards.

2.   Quality: Completeness (1–3 scale) SCORE = 2

·     Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. 

·   The data in Figs 3/4 could be strengthened by additional assays to measure inflammasome activation beyond IL-1β secretion and PI staining. Options include an IL-18 ELISA, LDH release assay, HMGB1 release assay, or caspase-1 cleavage assay (western blot).

·     Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. 

·   Beyond additional assays for inflammasome activation, we suggest that the authors could diversify the ‘priming’ treatments. For example, cells could be primed with LPS, poly I:C, or GM-CSF. Demonstrating CARD8-dependent inflammasome activation in conjunction with diverse stimuli will strengthen conclusions, and take away concerns about a TLR2-specific effect.

3. Quality: Reproducibility (1–3 scale) SCORE = 1

·     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

·   Sufficient biological replicates of all assays support the author’s conclusions.    

·     Is there sufficient raw data presented to assess the rigor of the analysis? 

·   Yes

·     Are methods for experimentation and analysis adequately outlined to permit reproducibility? 

·   Yes

·     If a ‘‘discovery’ dataset is used, has a ‘‘validation’ cohort been assessed and/or has the issue of false discovery been addressed?  

·   N/A

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5

·     Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? 

·   Yes         

·     Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? 

·     Yes

·     Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions.

·       The following statement stimulated a lot of discussion amongst the students, who were concerned that the study doesn’t really address pathogenesis: “Taken together, our work highlights how even minor, single amino acid changes can have dramatic, species-specific impacts on innate immune sensing and pathogenesis, and provides a model to explain, in part, the unique susceptibility of humans to HIV pathogenesis.”

·       Minor points: We recommend changing the color scheme of graphs in Fig 3 to enhance readability. This could be achieved by color-coding, with one color per group (e.g. HIV-1LAI in blue and HIV-1LAI-VSVG in yellow).

MORE SUBJECTIVE CRITERIA (IMPACT): 

1.   Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE= 2.5

A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] 

How big of an advance would you consider the findings to be if fully supported but not extended?

·       If properly supported by corroborating assays, these findings represent a significant advance in the field without further extension.

2.   Impact: Extensibility (1–4 or N/A scale) SCORE =   2.5

Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?  This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2)

·       The manuscript explores human vs. chimpanzee CARD8 and diverse lentiviruses. No further extension is provided to a bigger scheme.

·       We did wonder whether other non-lentiviral proteases could also cleave human CARD8. This would clearly add to our understanding of selective pressures that could have influenced CARD8 evolution in primates. Coronaviruses are mentioned in the discussion. Investigating the activity of coronavirus or enterovirus proteases against CARD8 homologs from different primates would be fascinating. Some clues may already exist in the following n-terminomics studies:

o    Jagdeo JM, Dufour A, Klein T, Solis N, Kleifeld O, Kizhakkedathu J, Luo H, Overall CM, Jan E. N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection. J Virol. 2018 Mar 28;92(8):e02211-17. doi: 10.1128/JVI.02211-17. PMID: 29437971; PMCID: PMC5874412.

o    Pablos I, Machado Y, de Jesus HCR, Mohamud Y, Kappelhoff R, Lindskog C, Vlok M, Bell PA, Butler GS, Grin PM, Cao QT, Nguyen JP, Solis N, Abbina S, Rut W, Vederas JC, Szekely L, Szakos A, Drag M, Kizhakkedathu JN, Mossman K, Hirota JA, Jan E, Luo H, Banerjee A, Overall CM. Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL^pro substrate degradome. Cell Rep. 2021 Oct 26;37(4):109892. doi: 10.1016/j.celrep.2021.109892. Epub 2021 Oct 9. PMID: 34672947; PMCID: PMC8501228.

o    Koudelka T, Boger J, Henkel A, Schönherr R, Krantz S, Fuchs S, Rodríguez E, Redecke L, Tholey A. N-Terminomics for the Identification of In Vitro Substrates and Cleavage Site Specificity of the SARS-CoV-2 Main Protease. Proteomics. 2021 Jan;21(2):e2000246. doi: 10.1002/pmic.202000246. Epub 2020 Nov 17. PMID: 33111431; PMCID: PMC7645863.

o    Luo SY, Moussa EW, Lopez-Orozco J, Felix-Lopez A, Ishida R, Fayad N, Gomez-Cardona E, Wang H, Wilson JA, Kumar A, Hobman TC, Julien O. Identification of Human Host Substrates of the SARS-CoV-2 M^pro and PL^proUsing Subtiligase N-Terminomics. ACS Infect Dis. 2023 Apr 14;9(4):749-761. doi: 10.1021/acsinfecdis.2c00458. Epub 2023 Apr 3. PMID: 37011043; PMCID: PMC10081575.

o    Bell PA, Overall CM. No Substrate Left behind-Mining of Shotgun Proteomics Datasets Rescues Evidence of Proteolysis by SARS-CoV-2 3CL^pro Main Protease. Int J Mol Sci. 2023 May 13;24(10):8723. doi: 10.3390/ijms24108723. PMID: 37240067; PMCID: PMC10218362.

Competing interests

The author declares that they have no competing interests.