The authors have declared that no competing interests exist.
TRIM5α is a key cross-species barrier to retroviral infection, with certain TRIM5 alleles conferring increased risk of HIV-1 infection in humans. TRIM5α is best known as a species-specific restriction factor that directly inhibits the viral life cycle. Additionally, it is also a pattern-recognition receptor (PRR) that activates inflammatory signaling. How TRIM5α carries out its multi-faceted actions in antiviral defense remains incompletely understood. Here, we show that proteins required for autophagy, a cellular self-digestion pathway, play an important role in TRIM5α’s function as a PRR. Genetic depletion of proteins involved in all stages of the autophagy pathway prevented TRIM5α-driven expression of NF-κB and AP1 responsive genes. One of these genes is the preeminent antiviral cytokine interferon β (IFN-β), whose TRIM5-dependent expression was lost in cells lacking the autophagy proteins ATG7, BECN1, and ULK1. Moreover, we found that the ability of TRIM5α to stimulate IFN-β expression in response to recognition of a TRIM5α-restricted HIV-1 capsid mutant (P90A) was abrogated in cells lacking autophagy factors. Stimulation of human macrophage-like cells with the P90A virus protected them against subsequent infection with an otherwise resistant wild type HIV-1 in a manner requiring TRIM5α, BECN1, and ULK1. Mechanistically, TRIM5α was attenuated in its ability to activate the kinase TAK1 in autophagy deficient cells, and both BECN1 and ATG7 contributed to the assembly of TRIM5α-TAK1 complexes. These data demonstrate a non-canonical role for the autophagy machinery in assembling antiviral signaling complexes and in establishing a TRIM5α-dependent antiviral state.
TRIM5α is an antiretroviral protein that employs multiple mechanisms to protect cells against infection. Previous studies have linked TRIM5α to autophagy, a cytoplasmic quality control pathway with numerous roles in immunity, raising the possibility that TRIM5α engages autophagy in antiviral defense. This concept has been controversial, since TRIM5α’s best-known role as a directly acting antiretroviral effector is autophagy independent. However, retroviral restriction is only one aspect of TRIM5α function. We demonstrate that autophagy is crucial to another TRIM5α action: its role as a pattern-recognition receptor. We show that autophagy machinery is required for TRIM5α to transduce antiviral signaling and to establish an antiviral state. Our data indicate that autophagy provides TRIM5α with a platform upon which to activate antiviral responses.
The selective pressure imposed by retroviral infection has shaped the human genome and has driven the evolution of proteins that function to protect host cells against retroviral infection [
These expanded functions of TRIM5 have started to emerge. For instance, TRIM5 can act as a pattern-recognition receptor for retroviral capsid, triggering the expression of NF-κB- and AP1-regulated immune genes (e.g. interferon α/β, IL-6) [
Additionally, we and others have reported multiple linkages between TRIM5 and the macroautophagy pathway [
TRIM5 has been shown to interact with proteins required for the upstream initiation of autophagy (ULK1, BECN1, ATG14, and AMBRA1), with mAtg8 conversion machinery (ATG5 and ATG16L), with all members of the mAtg8 protein family, with the autophagosome maturation factor UVRAG, and with the selective autophagy receptor p62 [
Here we show that the autophagy machinery strongly contributes to the ability of TRIM5 to act as a pro-inflammatory signaling molecule and for it to establish a cellular antiviral state in response to retroviral capsid detection. Our studies suggest that autophagy proteins are required to provide a scaffold for the interaction between TRIM5 and the kinase TAK1. Autophagy factor depletion attenuates the ability of TRIM5 to drive TAK1 activation and downstream signaling. Collectively, these studies suggest a novel, non-degradative role of the autophagy machinery in assembling active signaling complexes and uncover a role for autophagy in TRIM5-mediated antiviral responses.
TRIM5 over-expression in HEK293T cells drives activation of NF-κB and AP1 reporters in a manner requiring the kinase TAK1 and the E2 ubiquitin conjugating enzyme UBC13/UBE2N (
(
(
We next asked what other modules of the autophagy machinery are required for TRIM5-based NF-κB and AP1 transcription factor activation. The results of the ATG7 experiments above indicated a role for mAtg8 proteins, which we confirmed following GABARAP knockdown (Figs
(
We next tested whether autophagy factor knockdown could impact TRIM5-driven gene expression in PMA-differentiated THP1 macrophages, as macrophages are one of the first cell types that HIV-1 is likely to encounter following transmission. To this end, we generated polyclonal cell lines stably transduced with C-terminally tagged rhesus or human TRIM5 or with the protein tag alone (
(
TRIM5 can stimulate innate immune signaling in response to its recognition of restriction-sensitive retroviral cores [
(
In the experiments described above, we saw that HIV-1 CA P90A induced the expression of antiviral interferon β in an autophagy factor-dependent manner. We next asked whether this effect was sufficient to affect the outcome of infection with TRIM5-resistant viruses. To do this, we devised a scheme of sequential infections in which THP-1 macrophages are “primed” with human TRIM5-restricted single-cycle viruses prior to being “challenged” by GFP-expressing HIV-1 pseudovirions with wild type (TRIM5-resistant) capsid. The number of HIV-1 infected (GFP-positive) cells is then determined by high content imaging (
(
We next tested whether priming with HIV-1 CA P90A could attenuate WT HIV-1 infection in ULK1 and BECN1 knockout THP-1 cells. We did not pursue these experiments with the ATG7 knockout THP-1 cells since this cell line showed enhanced resistance to HIV-1 transduction even in the absence of priming (
Control or knockout THP-1 macrophages were primed with P90A (CrFK MOI 3) for 24 hours prior to infection with WT HIV-GFP (CrFK MOI 1) for two days. (
We next investigated the mechanism underlying how autophagy factors facilitate TRIM5-dependent signaling. The kinase TAK1 is reported to be activated immediately downstream of TRIM5 in response to TRIM5-generated K63-linked poly-ubiquitin chains [
(
We next considered a model in which the autophagy machinery helps scaffold TRIM5-TAK1 interactions. A prediction of this model would be that disrupting TRIM5’s ability to interact with the autophagy machinery would impact TRIM5 signaling. We previously identified two nearly adjacent LC3-interacting motifs (LIRs) in the TRIM5 coiled-coil domain: 187FEQL190 (LIR1) and 196WEESN200 (LIR2) [
To alleviate this ambiguity, we carried out experiments using LIR2-mutated TRIM5 (196WE ➔ AA; ΔLIR2) [
Our study has demonstrated that the autophagy machinery is essential for TRIM5 to carry out its function as a pattern recognition receptor for retroviral cores (
(
We found that the signaling induced by HIV-1 CA P90A infection could potently inhibit the ability of otherwise TRIM5-resistant HIV-1 and Sendai virus to infect THP-1 macrophages. Importantly, the antiviral state established in response to HIV-1 CA P90A treatment required TRIM5 and the autophagy factors BECN1 and ULK1 (Figs
While our STR studies were not intended to mimic a clinically relevant setting, our results may provide an answer as to how human TRIM5, which has historically been considered ineffective as an HIV-1 restriction factor, can confer protection against HIV-1 infection risk in people [
How does the autophagy machinery impact TRIM5 signaling? The loss of autophagy proteins impacts both TRIM5-mediated NF-κB and AP1 activation, and thus we reasoned that autophagy proteins must be acting on the level of TAK1 since this kinase functions as a signaling node controlling both of these transcription factors. Accordingly, our studies demonstrated that the loss of autophagy factors ATG7, BECN1 or ULK1 reduced the ability of TRIM5 to promote TAK1 activation (
One surprising finding of our study was that all stages of the autophagy pathway, including the later degradative steps, appear to be important for TRIM5-dependent signaling. We used luciferase expression reporter assays to screen different modules of the autophagy machinery for roles in TRIM5-induced signaling. We found that factors acting in autophagy initiation (ULK1 and BECN1), substrate selectivity (p62), autophagosome membrane elongation (ATG7 and GABARAP), autophagosome closure (CHMP2A), and autophagosome-lysosome fusion (VAMP8 and SNAP29) are all important for NF-κB and AP1 transcriptional activation in cells transiently expressing TRIM5. These findings strongly suggest that the TRIM5-TAK1 signaling axis requires full autophagy rather than single components of the autophagy machinery to assemble TRIM5-TAK1 complexes. However, why autophagosome-lysosome fusion mediated by VAMP8 and SNAP29 is important for TRIM5 signaling is still an open question. Our data indicated that inhibition of lysosomal proteases mildly attenuated the ability of TRIM5 expression to stimulate NF-κB activation, thus one possible answer could be that autophagy degrades or deactivates an as-yet unidentified negative regulator or TRIM5-TAK1 signaling; with one or more of the ~100 deubiquitinating enzymes being likely candidates.
In conclusion, our study shows that the autophagy machinery provides a signaling scaffold allowing TRIM5 to initiate an antiviral state that is protective against HIV-1. This explains the extensive connections between TRIM5 and autophagy and establishes a novel non-canonical role for the autophagy machinery in assembling TRIM5-TAK1 complexes. Finally, TRIM5 is one member of the large TRIM protein family. A very high percentage of the ~70 TRIMs in human genome act in regulating inflammatory signaling and antiviral defense and are increasingly linked to autophagy [
HEK293T, HeLa, THP-1 and CrFK cells were obtained from the American Type Culture Collection (ATCC). HEK293T, HeLa and CrFK cells were grown in Dulbecco’s modified Eagle’s medium (Life technologies,11965126) supplemented with 10% fetal bovine serum (FBS, Life technologies, 26140–079), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a 5% CO2 atmosphere. HeLa cells stably expressing HA-tagged RhTRIM5α were obtained from NIH AIDS reagents and were maintained in the above media supplemented with 1 μg/ml puromycin. THP-1 cells were maintained in RPMI 1640 (Corning, 10-040-CV) containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose (Corning, 25-037-CL), 10 mM HEPES (ThermoFisher, 15630080) and 1.0 mM sodium pyruvate (ThermoFisher, 11360–070), 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. THP-1 cells were differentiated with 50 ng/mL PMA (Sigma, P8139) for 24h, washed and incubated in complete medium for 48h before experimental use. APEX2-V5, RhTRIM5-APEX2-V5 and HuTRIM5-APEX2-V5 stable overexpression in WT or KO THP-1 backgrounds was achieved by viral transduction followed by 14–21 days of culturing in medium containing the selective antibiotic (1 μg/ml puromycin) before stable integration of the target gene was confirmed by western blotting. ULK1, BECN1, ATG7 and TRIM5 knockout (KO) HEK293T and THP1 cells were generated by transduction with lentiCRISPRv2-based lentiviruses followed by 2–4 weeks of culturing in medium containing the selectable marker (1 μg/ml puromycin for ULK1, BECN1, ATG7 KO cells or 200 μg/ml hygromycin for TRIM5 KO cells). Knockout lines were confirmed by immunoblot.
Viral particles for the generation of stable overexpressed cell lines were produced by co-transfection of pLEX_307 (a gift from David Root, Addgene plasmid # 41392) containing the target gene, psPAX2 and pMD2.G at the ratio of 1:1:1 in HEK293T cells using ProFection Mammalian Transfection System (Promega, E1200), medium was changed 16h post transfection and virus containing supernatant was harvested 48h later, clarified by centrifuging for 5 min at 1200 rpm, 0.45 μm-filtered (Millipore, SE1M003M00), diluted with full medium at 1:1 ratio and used to transduce target cells for 48h in 6 cm dishes.
Viral particles for the generation of knockout cell lines were produced by transfecting HEK293T cells with a lentiviral vector, lentiCRISPRv2 carrying both Cas9 enzyme and a guide RNA targeting specific gene together with the packaging plasmids psPAX2 and pMD2.G at the ratio of 10 μg, 10 μg and 10 μg/10 cm dish. Lentiviral particles were harvested from supernatants as mentioned above and HEK293T, Huh7, and THP-1 cells were infected in the presence of polybrene for 48h in 6 cm dishes.
In this study, all virus infection was performed using VSV-G pseudotyped single-cycle HIV-1 or N-MLV produced by transfection of HEK293T cells. 24h before transfection, 2.2×106 HEK293T cells were seeded in 10 cm dishes. Two-part single cycle VSV-G-pseudotyped HIV-1 (NL43 strain) was collected from the supernatants of HEK293T cells transfected with plasmids encoding VSV-G and HIV-1 lacking the Env gene (Env-, VPR-, Nef+, IRES-GFP) at 1:2 ratios. Three part, single cycle VSV-G-pseudotyped N-MLV, HIV1 WT and recombinant HIV-1 strain (HIV-1 CA P90A) were produced by co-transfecting 15 μg lentivector genome plasmid (pLVX-mcherry), 10 μg gag-pol plasmid (pCIG3-N for N-MLV, WT and P90A HIV-1 capsid[
pDest40-APEX2-V5 and pLEX_307-APEX2-V5; pDest40-RhTRIM5-APEX2-V5 and pLEX_307- RhTRIM5-APEX2-V5; pDest40-HuTRIM5-APEX2-V5 and pLEX_307-HuTRIM5-APEX2-V5; LIR2 mutant pDest40-GFP-RhTRIM5 were generated using Gateway recombination cloning. First, they were PCR amplified from available cDNA clones and recombined into pDONR221 using the BP reaction (Life Technologies, 11789–020) prior to being recombined into expression plasmids by LR cloning (Life Technologies, 11791–020). Plasmid constructs were verified by DNA sequencing. The AP1 luciferase reporter plasmid was a gift from Alexander Dent (Addgene plasmid #40342; 3XAP1pGL3), the NF-κB luciferase reporter was purchased from Promega (#E8491) and the Renilla luciferase plasmid (pRL-SV40, Addgene plasmid #27163) was a gift from Ron Prywes. All other plasmids have been previously published [
The following primary antibodies were used: Beclin1 (Cell Signaling, 3495S), ATG7 (Cell Signaling, 8558S), ULK1 (Cell Signaling, 8054S), p62 (BD, 610833), UBC13 (Abcam, 25885), GABARAP (Cell Signaling, 13733S), TFEB (Cell Signaling, 37785S), CHMP2A (Proteintech, 10477-1-AP), VAMP8 (Abcam #76021), SNAP29 (Abcam #138500), V5 (Cell Signaling, 13202S), TRIM5 (Abcam, Ab59000; Cell Signaling #14326), phospho-TAK1 (Cell Signaling, 4508S), FLAG (Sigma, F1804), GFP (Abcam, Ab290), HA (Abcam, Ab9110), c-Myc (Santa Cruz, 40) and actin (Santa Cruz, 58673). Secondary antibodies used were fluorescently conjugated goat anti-mouse (LI-COR, 925–68020) and goat anti-rabbit (LI-COR, 925–32210), HRP-conjugated goat anti-mouse (Bio-Rad, 1721011) and goat anti-rabbit (Bio-Rad, 1721019) or Clean-Blot HRP (ThermoFisher, 21230). Nuclease free water (Dharmacon, B-003000-WB-100), 5X siRNA buffer (Dharmacon, B-002000-UB-100), Opti-MEM Reduced Serum Medium (ThermoFisher, 31985070), RIPA lysis buffer (ThermoFisher, 89901), phenylmethylsulfonyl fluoride (PMSF, Sigma, 93482, 1 mM), protease inhibitor cocktails (ROCHE, 11836170001), BSA (Fisher Scientific, BP 1600–1), Puromycin (Sigma, P8833), Polybrene (EMD Millipore, TR-1003-G, 10 μg/ml), Hygromycin (Corning, 30-240-CR), MG132 (Selleckchem, S21619, 0.2 μM), IP lysis buffer (ThermoFisher, 87788), phosphatase inhibitor cocktails (ROCHE, 04906845001), Dynabeads Protein G (ThermoFisher, 10004D) and Restore PLUS Western blot stripping buffer (ThermoFisher, 46430). NF-κB and AP1 inhibitors (BAY 11–7085 and SP600125) were purchased from Enzo Life Sciences. 3-methyladenine, e64d, and pepstatin A were purchased from Sigma.
For siRNA mediated knockdown experiments, HEK293T cells (1.2×106 cells/6cm dish) were reverse transfected with non-targeting or the indicated siRNA using Lipofectamine RNAiMAX, after 24h cells were harvested and reseeded in 96 well plate (20000 cells/well in 100 μl tissue culture medium). For other luciferase experiments, 20000 HEK293T cells were plated in each well of a 96 well plate 24h prior to transfection. Cells were transfected using 0.25 μL Lipofectamine 2000 per well, with 10 ng of the
Total RNA was extracted with RNeasy Plus Mini kit (Qiagen, 74104) and first strand cDNA was synthesized using random hexamers as primers (High-Capacity cDNA Reverse Transcriptase Kit, Thermo Fisher, 4368814), in accordance with manufacturer’s instructions. qPCR reactions were performed with TaqMan Gene Expression Assays (Thermo Fisher) for, IFNB1 (Hs01077958_s1), NLRP1 (Hs00248187_m1), IL6 (Hs00985639_m1), IL10 (Hs00961622_m1), SEV (Mr04269880_mr), and 18S rRNA (Fn0464250_s1) was used as a house keeping gene for normalization. The qPCR assays were run on the StepOnePlus Real-Time PCR System (Applied Biosystems).
For CrFK, Huh7, and HeLa cells, 8000 cells were seeded per well in a 96 well plate, 24h prior to virus challenge. Media containing VSV-G pseudotyped lentiviral vectors expressing HIV1 WT or HIV1-P90A or N-MLV was added to infect cells in a total volume of 100 μL in the presence of polybrene. For THP1 cells, 50000 cells were seeded in each well of a 96 well plate in a culture medium containing 50 ng/mL PMA for 24h, then washed and placed in normal medium. After 48h, differentiated THP-1 cells were infected with the virus using MOI specified in figure legends. After infection, cells were first incubated at 4°C for 1h to allow the virus to bind. Free virus was then removed by washing and cells were incubated in complete medium. 48h post infection, cells were fixed, stained with Hoechst 33342 and the fraction of transduced cells showing fluorescent protein positivity was determined by high content imaging and analysis. Culture supernatant from un-transfected HEK293T cells was diluted in complete media and used for mock infections. Infection was determined by measuring the percentage of GFP or mCherry positive cells. All the infections were repeated multiple times (>3).
THP1 control and the different THP1 KO cells were seeded into 96 well plates at a density of 50000 cells/well, differentiated with PMA, then primed with VSV-G pseudotyped HIV-mCherry (at a CrFK MOI of 3) or VSV-G pseudotyped HIV-mCherry CA P90A (at a CrFK MOI of 3) or VSV-G pseudotyped N-MLV-mCherry (at a CrFK MOI of 0.5), 24h later media was removed, washed and challenged with different dilutions of VSV-G pseudotyped HIV1-WT-GFP tagged (CrFK MOI of 2 was used as the starting dilution). For most experiments, we challenged cells with a CrFK MOI of 1. 48h post second virus infection, cells were fixed, stained with Hoechst 33342 and percentage of GFP positive cells were determined using high content imaging. Where indicated, cells were treated with 0.2 μM MG132 during the virus priming.
Sendai virus (formerly Parainfluenza Virus 1, Sendai) was obtained through BEI Resources, NIAID, NIH: NR-3227. Cells were inoculated with virus in RPMI media at a concentration of 50 hemagglutination units (HAU)/mL. To quantify experiments, RNA was collected in Trizol Reagent (Invitrogen catalog# 15596026) and purified with the Zymo Research Direct-zol RNA Miniprep Kit (catalog# R2052). Total RNA concentration was normalized and cDNA synthesis performed with Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (catalog# 43-688-14).
All high content experiments were performed in 96-well plate format. After the indicated treatments, cells were fixed with 4% paraformaldehyde for 10 min, washed twice with 1X PBS and stained with the nuclear stain Hoechst 33342. Nuclear staining was used for autofocus and to automatically define cellular outlines. High content imaging and analysis were performed using a Cellomics CellInsight CX7 scanner and iDEV software (Thermo); > 2000 cells were analyzed per well, and 10–12 wells of the 96 well plate were analyzed per sample. Transduced cells were automatically identified based on having above background fluorescent protein signal in the nucleus. All data acquisition and analysis were computer driven and independent of human operators.
Most immunoprecipitation and immunoblots were as described [
HeLa cells stably expressing HA-tagged RhTRIM5α were plated onto glass coverslips in 12 well plates prior to being transfected with the plasmids indicated in figures. Samples were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% saponin in 3% BSA and blocked with 3% BSA in PBS. Intracellular targets were then stained with primary antibodies according to the manufacturer’s recommendation, washed three times with PBS, followed by incubation with Alexa Fluor conjugated secondary antibodies for 1 h at room temperature. Coverslips were mounted using ProLong Diamond Antifade Mountant (Invitrogen, P36970). Images were acquired using a Zeiss LSM800 microscope and analyzed using Zen2 software (Zeiss) and deconvolved using Huygens Essential software (Scientific Volume Imaging).
Data are expressed as means ± SEM (n>3). Data were analyzed with unpaired two-tailed t-tests or ANOVA with Bonferroni post hoc analysis. Statistical significance is defined as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
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We thank Dr. Edward Campbell (Loyola University Chicago) for providing plasmids and for helpful discussions. We thank Dr. Kiran Bhaskar (University of New Mexico) for the use of his qPCR instrument and Drs. Diane Lidke and Suresh Kumar (University of New Mexico) for critically reading the manuscript.
Dear Dr. Mandell,
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Reviewer's Responses to Questions
Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.
Reviewer #1: TRIM5a is one of the best-studied Tripartite motif proteins. It is well-recognized as a Lentiviral restriction factor. While it is known that TRIM5a recognizes capsids in a species-specific manner, and induces premature uncoating, the consequences and mode of signaling induced by TRIM5a are less clear. Particularly the involvement of autophagy has been under debate.
It has been proposed that TRIM5a may serves as an innate immune receptor. Although the pattern recognized by this factor may be really specialized to qualify as a pattern recognition receptor.
In this manuscript Saha and co-workers show that TRIM5-dependent cytokine/Nf-kB induction requires core autophagy proteins such as ATG7 or beclin-1. Pre-stimulation of cells with TRIM5a-recognized capsid renders the cells less susceptible towards a subsequent HIV-1 infection.
Most experiments are well conducted and controlled. The findings presented would be novel and an interesting aspect of signaling regulation by autophagy. However, there are a few things which may need some clarification.
Reviewer #2: This paper describes a non-canonical role for the cellular autophagy machinery in TRIM5 mediated innate signalling. I think this is a very important result. However, the results section of the paper is at times rather hard to follow with some data seemingly randomly assigned to Figs or Suppl Figs- did a limit in the number of figures play a role? Why for example did parts A and B of Fig 2 appear there when the experiments reported were performed in HEK cells, not macrophages as in the figure title. I wonder whether some of the normalized data would be better presented as tables. I would also note that improved quantification would be very helpful in assessing many of the protein blots.
Reviewer #3: This is an nice study that is comprehensive and well controlled. The paper is well-written and makes important conceptual advances in the field of antiviral signaling pathways, by enhancing our understanding of how autophagy contributes to the ability of TRIM5a to promote the IFN response.
In my opinion, this work will be of general interest to a broad audience in molecular biology and cell biology.
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Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".
Reviewer #1: - There is a control missing in P90A capsid pre-stimulation assay. How do capsids modulate the IFN-b response and a subsequent infection that are not recognized by TRIM5a? Please include priming with a non TRIM5-sensitive virus in Fig. 4.
- I wonder whether the impact on Nf-kB signaling is a common feature of that signaling pathways or unique for TRIM5a-dependent induction? The authors should include controls in the assays in Fig.1, e.g. overexpression of IKKa/b or other means to stimulate an Nf-kB response?
- In Fig. 2D, the IFNb levels induced by RhTRIM5 and HuTRIM5 are the same, however huTRIM5 has no effect on virus replication. Would that mean that the signaling of TRIM5a does not impact the virus?
- Please include TRIM5a KO in Fig. 3B and C.
- Fig. 6D and E: The IP levels of TAK1 and phosphor-TAK1 visually correlate with Input FLAG-TAK1 or GFP-TRIM5, please quantify these IPs and normalize to the respective controls, to strengthen the conclusion.
Reviewer #2: (No Response)
Reviewer #3: I offer a few minor suggestions for the authors to consider:
1) Potential candidates for the regulation of TAK1 activity by TRIM5a could be TAB2/TAB3. The authors should test in their experimental settings if TAB2/TAB3 levels and/or their interaction with TAK1 are altered by downregulating the expression of autophagy genes.
2) It remains unclear if, besides autophagy proteins, the autophagic degradative activity is also required for the regulation of NF-kB signaling. It would be interesting to test if lysosomal inhibitors affect IFN induction by TRIM5a
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Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.
Reviewer #1: - Please replace Fig. 2C with a more quantitative FACS assay
- Fig 5 C and D would benefit from a more transparent presentation of the data
- Fig 6 A has to be quantified: phosphor-Tak vs Tak levels
- Fig 6B and C, all the single overexpressions and stains need to be shown. Please quantify the co-localisation.
- Fig. 6G: This dataset is relatively weak, although it is explained why. However, are there any other mutants of TRIM5a which may be more suitable to show that autophagy is required for Nf-kB signaling?
- Processing of signaling compounds may be important. But also degradation of NIK (and other negative regulators of Nf-kB signaling) as a major factor (besides DUBs), could be experimentally checked or addressed in the discussion.
- Can the authors include a model of their suggested mechanism?
Reviewer #2: L139ff. It appears that BECN1, ATG7, ULK1 knockdowns/outs have a greater effect on NF-KB than AP-1. Is this true? If so what does it mean?
L141. Requiring
L183ff. There appear to be differences between hu and rhTRIM5, especially in NLRP1 responses. Do you think these are important? I wonder whether it would be worth testing RBCC/PrySpry chimeras?
L202ff. Is over-expression in some way similar to CA binding? Could it be that hexameric TRIM5 is needed for TAK1 activation and that this occurs at a certain rate naturally and is enhanced by over-expression or virus binding?
No reference in text to Fig S3A
L455ff. Is the preparation of deltaLIR1/2 described here?
Fig 2. What is Scr siRNA? Is Beclin1 siRNA the same as BECN1 siRNA in Fig 1?
Fig 4. In Fig 3A CrFK moi-3 gave c.30% infected cells in 4C CrFK a moi of 2 gave c.50% infection. Needs an explanation. Also the differences going from moi of 2 to 1 in Figs 4C,D
Fig 6A. Does ATG7 really increase amount of p-TAK1 in GFP control?
Fig 6B, C. Very hard to make out important details
Fig 6E. Is GFP-TRIM supposed to go down in IP but not in input?
Fig 6F. Why are there two sets of FLAG-TAK1 samples?
Fig S3A. Is it fair to say that on a per cell basis HIV P90A is more efficient that poly(I:C)?
Fig S5B. See labels GFP-RhTRIM5 and GFP-TRIM5. Is the Rh a mistake? Are all these experiments done with wt and deleted human TRIM5?
Reviewer #3: None
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PLOS Pathogens
Michael Malim
Editor-in-Chief
PLOS Pathogens
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Reviewer Comments (if any, and for reference):
Dear Dr. Mandell,
We are delighted to inform you that your manuscript, "A non-canonical role for the autophagy machinery in anti-retroviral signaling mediated by TRIM5α.," has been formally accepted for publication in PLOS Pathogens.
We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.
The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.
Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.
Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.
Best regards,
Kasturi Haldar
Editor-in-Chief
PLOS Pathogens
Michael Malim
Editor-in-Chief
PLOS Pathogens