Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
In this manuscript, the authors employ a combined proteomic and genetic approach to identify the glycoprotein QC factor malectin as an important protein involved in promoting coronavirus infection. Using proteomic approaches, they show that the non-structural protein NSP2 and malectin interact in the absence of viral infection, but not in the presence of viral infection. However, both NSP2 and malectin engage the OST complex during viral infection, with malectin also showing reduced interactions with other glycoprotein QC proteins. Malectin KD reduce replication of coronaviruses, including SARS-COV2. Collectively, these results identify Malectin as a glycoprotein QC protein involved in regulating coronavirus replication that could potentially be targeted to mitigate coronavirus replication.
Overall, the experiments described appear well performed and the interpretations generally reflect the results. Moreover, this work identifies Malectin as an important pro-viral protein whose activity could potentially be therapeutically targeted for the broad treatment of coronavirus infection. However, there are some weaknesses in the work that, if addressed, would improve the impact of the manuscript.
Notably, the mechanism by which malectin regulates viral replication is not well described. It is clear from the work that malectin is a pro-viral protein in the work presented, but the mechanistic basis of this activity is not pursued. Some potential mechanisms are proposed in the discussion, but the manuscript would be strengthened if additional insight was included. For example, does the UPR activated to higher levels in infected cells depleted of malectin? Do glycosylation patterns of viral (or non-viral) proteins change in malectindepleted cells? Additional insight into this specific question would significantly improve the manuscript.
We concur with the reviewer that the mechanism by which Malectin regulates viral replication is an important point to elucidate further. Our proteomics data were able to offer additional insight into the questions posed here. We examined the upregulation of protein markers of the UPR and other stress response pathways in cells depleted of MLEC (Fig. S15D). We find that the UPR pathways are moderately but insignificantly upregulated, while the Heat Shock Factor 1 (HSF1) pathway is moderately and significantly upregulated. The fold change increase of these marker proteins are relatively small, so while upregulation of this pathway may contribute to the suppression of CoV replication, it may not fully explain the phenotype.
In addition, to address the second question, we compared the glycosylation patterns of endogenous proteins in MLEC-KD cells (Fig. S15E-G). We found that there is a small increase in abundance of glycopeptides associated with LAMP2, SERPHINH1, RDX, RPL3/5, CADM4, and ITGB1, however these fold changes are small and tested to be insignificant. These results indicate there is relatively little modification of endogenous glycoproteins upon MLEC-depletion. These findings support a more direct role for MLEC in regulating viral replication.
We added the following section to the manuscript text to discuss these results:
“In uninfected cells, MLEC KD leads to relatively little proteome-wide changes, with MLEC being the only protein significantly downregulated and no other proteins significantly upregulated, supporting the specificity of MLEC KD in MHV suppression (Fig. S15C). To determine whether MLEC KD alters general host proteostasis, we further examined the levels of protein markers of stress pathways based on previous gene pathway definitions(Davies et al., 2023; Grandjean et al., 2019; Shoulders et al., 2013) (Fig. S15D). We find that there are modest but significant increases in protein levels associated with the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response (UPR) pathways are largely unmodified.
We also probed the effect of MLEC KD on endogenous protein glycosylation. We find that there is only a small increase in abundance of glycopeptides, including those associated with the ribosome (Rpl3, Rpl5), a cytoskeletal protein (Rdx), the integrin Itgb1, and the ER-resident chaperone Serphinh1 (Fig. S15E-G).”
“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). In addition, there are only minor increases in endogenous glycopeptide levels (Fig. S15E-G). Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”
Further, the evidence for increased interactions between OST and malectin during viral infection is fairly weak, despite being a major talking point throughout the manuscript. The reduced interactions between malectin and other glycoproteostasis QC factors is evident, but the increased interactions with OST are not well supported. I'd recommend backing off on this point throughout the text, instead, continuing to highlight the reduced interactions.
We agree that the fold change increase of OST interactions with malectin are small compared to the fold change decrease of other glycoproteostasis factors We have modified the text to less emphasize this point and instead highlight the reduced interactions:
“Further, MHV infection retains the association of MLEC with the OST complex while titrating off other interactors, potentially leading to more efficient glycoprotein biogenesis.”
I was also curious as to why non-structural proteins, nsp2 and nsp4, showed robust interactions with host proteins localized to both the ER and mitochondria? Do these proteins localize to different organelles or do these interactions reflect some other type of dysregulation? It would be useful to provide a bit of speculation on this point.
We also find these ER and mitochondrial protein interactions curious, which we initially reported on (Davies, Almasy et al. 2020 ACS Infectious Diseases). In this prior report, we found that when expressed in HEK293T cells, SARS-CoV-2 nsp2 and nsp4 have partial localization to mitochondrial-associated ER membranes (MAMs), as determined by subcellular fractionation. Given that malectin has also been shown to have MAMs localization (Carreras-Sureda, et al. 2019 Nature Cell Biology), we have added additional text in the Discussion to speculate on this point:
“Additionally, MLEC has also been shown to localize to ER-mitochondria contact sites (MAMs)(Carreras-Sureda et al., 2019), which regulate mitochondrial bioenergetics. We have previously shown that SARS-CoV-2 nsp2 and nsp4 can partially localize to MAMs(Davies et al., 2020), so these viral proteins may also dysregulate MLEC and MAMs activity to promote infection.”
Again, the overall identification of malectin as a pro-viral protein involved in the replication of multiple different coronaviruses is interesting and important, but additional insights into the mechanism of this activity would strengthen the overall impact of this work.
Thank you for this endorsement. We hope the additional analyses and discussion points in the revised manuscript further homed in on a direct mechanistic function for MLEC in modulating viral replication.
Reviewer #2 (Public Review):
Summary:
A strong case is presented to establish that the endoplasmic reticulum carbohydrate binding protein malectin is an important factor for coronavirus propagation. Malectin was identified as a coronavirus nsp2 protein interactor using quantitative proteomics and its importance in the viral life cycle was supported by using a functional genetic screen and viral assays. Malectin binds diglucosylated proteins, an early glycoform thought to transiently exist on nascent chains shortly after translation and translocation; yet a role for malectin has previously been proposed in later quality control decisions and degradation targeting. These two observations have been difficult to reconcile temporally. In agreement with results from the Locher lab, the malectininteractome shown here includes a number of subunits of the oligosaccharyltransferase complex (OST). These results place malectin in close proximity to both the co-translational (STT3A or OST-A) and post-translational (STT3B or OST-B) complexes. It follows that malectin knockdown was associated with coronavirus Spike protein hypoglycosylation.
Strengths:
Strengths include using multiple viruses to identify interactors of nsp2 and quantitative proteomics along with multiple viral assays to monitor the viral life cycle.
Weaknesses:
Malectin knockdown was shown to be associated with Spike protein hypoglycosylation. This was further supported by malectin interactions with the OSTs. However, no specific role of malectin in glycosylation was discussed or proposed.
We have emphasized our hypotheses on this point in the discussion and added a summary figure to highlight the specific role of malectin.
Given the likelihood that malectin plays a role in the glycosylation of heavily glycosylated proteins like Spike, it is unfortunate that only 5 glycosites on Spike were identified using the MS deamidation assay when Spike has a large number of glycans (~22 sites). The mass spec data set would also include endogenous proteins. Were any heavily glycosylated endogenous proteins hypoglycosylated in the MS analysis in Fig 5D?
Thank you for this suggestion. We compared the glycosylation patterns of endogenous proteins in MLEC-KD cells (Fig. S15E-G). We found that there is a small increase in abundance of glycopeptides associated with LAMP2, SERPHINH1, RDX, RPL3/5, CADM4, and ITGB1, however these fold changes are small and tested insignificant. These results indicate there is relatively little modification of endogenous glycoproteins upon MLEC-depletion. We added the following sections:
“We also probed the effect of MLEC KD on endogenous protein glycosylation. We find that there is only a small increase in abundance of glycopeptides, including those associated with the ribosome (Rpl3, Rpl5), a cytoskeletal protein (Rdx), the integrin Itgb1, and the ER-resident chaperone Serphinh1 (Fig. S15E-G).”
“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). In addition, there are only minor increases in endogenous glycopeptide levels (Fig. S15E-G). Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”
The inclusion of the nsp4 interactome and its partial characterization is a distraction from the storyline that focuses on malectin and nsp2.
We believe the nsp4 comparative interactome and functional genomics data offers a rich resource for further functional investigation by others, if made public. While we found the malectin and nsp2 storyline the most compelling to pursue, we believe the inclusion of the nsp4 data strengthens the overall approach, in agreement with Reviewer #3’s comments.
Reviewer #3 (Public Review):
Summary:
In this study, Davies and Plate set out to discover conserved host interactors of coronavirus non-structural proteins (Nsp). They used 293T cells to ectopically express flag-tagged Nsp2 and Nsp4 from five human and mouse coronaviruses, including SARS-CoV-1 and 2, and analyzed their interaction with host proteins by affinity purification mass-spectrometry (AP-MS). To confirm whether such interactors play a role in coronavirus infection, the authors measured the effects of individual knockdowns on replication of murine hepatitis virus (MHV) in mouse Delayed Brain Tumor cells. Using this approach, they identified a previously undescribed interactor of Nsp2, Malectin (Mlec), which is involved in glycoprotein processing and shows a potent pro-viral function in both MHV and SARS-CoV-2. Although the authors were unable to confirm this interaction in MHVinfected cells, they show that infection remodels many other Mlec interactions, recruiting it to the ER complex that catalyzes protein glycosylation (OST). Mlec knockdown reduced viral RNA and protein levels during MHV infection, although such effects were not limited to specific viral proteins. However, knockdown reduced the levels of five viral glycopeptides that map to Spike protein, suggesting it may be affected by Mlec.
Strengths:
This is an elegant study that uses a state-of-the-art quantitative proteomic approach to identify host proteins that play critical roles in viral infection. Instead of focusing on a single protein from a single virus, it compares the interactomes of two viral proteins from five related viruses, generating a high confidence dataset. The functional follow-ups using multiple live and reporter viruses, including MHV and CoV2 variants, convincingly depict a pro-viral role for Mlec, a protein not previously implicated in coronavirus biology.
Weaknesses:
Although a commonly used approach, AP-MS of ectopically expressed viral proteins may not accurately capture infection-related interactions. The authors observed Mlec-Nsp2 interactions in transfected 293T cells (1C) but were unable to reproduce those in mouse cells infected with MHV (3C). EIF4E2/GIGYF2, two bonafide interactors of CoV2 Nsp2 from previous studies, are listed as depleted compared to negative controls (S1D). Most other CoV2 Nsp2 interactors are also depleted by the same analysis (S1D). Previously reported MERS Nsp2 interactors, including ASCC1 and TCF25, are also not detected (S1D). Furthermore, although GIGYF2 was not identified as an interactor of MHV Nsp2/4 in human cells (S1D), its knockdown in mouse cells reduced MHV titers about 1000 fold (S4). The authors should attempt to explain these discrepancies.
We acknowledge these limitations in AP-MS from ectopically expressed viral proteins and have addressed these discrepancies with further elaboration in the text:
“A limitation of our study is the initial overexpression of individual proteins for AP-MS, in which we find some variation between our data with other AP-MS studies. We sought to overcome these variations by focusing on conserved interactors and testing interactions in a live infection context.”
“We also found GIGYF2-KD strongly suppressed MHV infection, despite GIGYF2 not interacting with MHV nsp2 (Fig. S1D), highlighting the importance of proteostasis factors in infection regardless of direct PPIs.”
More importantly, the authors were unable to establish a direct link between Mlec and the biogenesis of any viral or host proteins, by mass-spectrometry or otherwise. Although it is clear that Mlec promotes coronavirus infection, the mechanism remains unclear. Its knockdown does not affect the proteome composition of uninfected cells (S15B), suggesting it is not required for proteome maintenance under normal conditions. The only viral glycopeptides detected during MHV infection originated from Spike (5D), although other viral proteins are also known to be glycosylated. Cells depleted for Mlec produce ~4-fold less Spike protein (4E) but no more than 2-fold less glycosylated spike peptides (5D), compounding the interpretation of Mlec effects on viral protein biogenesis. Furthermore, Spike is not essential for the pro-viral role of Mlec, given that Mlec knockdown reduces replication of SARS-CoV-2 replicons that express all viral proteins except for Spike (6A/B).
Thank you, these are all important points. We have acknowledged these compounding factors in the Discussion:
“Concurrently, knockdown of MLEC leads to impediment of nsp production and aberrant glycosylation of other viral proteins like Spike, though it should be noted that the decrease in Spike glycopeptides is compounded by the overall decrease in Spike protein. Given that MLEC is pro-viral in a SARS-CoV-2 replicon model lacking Spike (Fig. 6), MLEC can promote CoV replication independent of Spike production.”
Any of the observed effects on viral protein levels could be secondary to multiple other processes.Interventions that delay infection for any reason could lead to an imbalance of viral protein levels because Spike and other structural proteins are produced at a much higher rate than non-structural proteins due to the higher abundance of their cognate subgenomic RNAs. Similarly, the observation that Mlec depletion attenuates MHV-mediated changes to the host proteome (S15C/D) can also be attributed to indirect effects on viral replication, regardless of glycoprotein processing. In the discussion, the authors acknowledge that Mlec may indirectly affect infection through modulation of replication complex formation or ER stress, but do not offer any supporting evidence. Interestingly, plant homologs of Mlec are implicated in innate immunity, favoring a more global role for Mlec in mammalian coronavirus infections.
We examined the upregulation of protein markers of the UPR and other stress response pathways in cells depleted of MLEC (Fig. S15D). We find that the UPR pathways are moderately but insignificantly upregulated, while the Heat Shock Factor 1 (HSF1) pathway is moderately and significantly upregulated. The fold change increase of these marker proteins are relatively small, so while upregulation of this pathway may contribute to the suppression of CoV replication, it may not fully explain the phenotype. Please all see similar points brought up by reviewer 1 (comment 1). We added the following section to the manuscript text to discuss these results:
“In uninfected cells, MLEC KD leads to relatively little proteome-wide changes, with MLEC being the only protein significantly downregulated and no other proteins significantly upregulated, supporting the specificity of MLEC KD in MHV suppression (Fig. S15C). To determine whether MLEC KD alters general host proteostasis, we further examined the levels of protein markers of stress pathways based on previous gene pathway definitions(Davies et al., 2023; Grandjean et al., 2019; Shoulders et al., 2013) (Fig. S15D). We find that there are modest but significant increases in protein levels associated with the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response (UPR) pathways are largely unmodified.
“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). […] Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”
Finally, the observation that both Nsp2 (3C) and Mlec (3E/F) are recruited to the OST complex during MHV infection neither support nor refute any of these alternate hypotheses, given that Mlec is known to interact with OST in uninfected cells and that Nsp2 may interact with OST as part of the full length unprocessed Orf1a, as it co-translationally translocates into the ER. Therefore, the main claims about the role of Mlec in coronavirus protein biogenesis are only partially supported.
We have acknowledged this point in the Discussion.
“We find that nsp2 interacts with several OST complex members, including DDOST, STT3A, and RPN1, though whether this is as part of the uncleaved Orf1a polyprotein during co-translational ER translocation or as an individual protein is unclear.”
Reviewer #2 (Recommendations For The Authors):
What is the proof that MLEC is a type I membrane protein? If it is strictly sequence analysis, this conclusion should be tapered in the text.
Our response: We have added appropriate evidence on the biochemical characterization of MLEC topology from Galli et al., 2011, and cryo-EM structural characterization by Ramírez et al., 2019.
“As it was surprising that nsp2, a non-glycosylated, cytoplasmic protein, would interact with MLEC, an integral ER membrane protein with a short two amino acid cytoplasmic tail(Galli et al., 2011; Ramírez et al., 2019), we assessed a broader genetic interaction between nsp2 and MLEC.”
Validation of some of the nsp2 and malectin interactome components by pulldowns should be included.
Our response: The interactions between nsp2 and Ddost, Stt3A, and Rpn1 passed a stringent confidence filter in our AP-MS experiment (Fig. 3C) based on several replication. For this reason, we do not believe additional validation by Western blotting will offer much useful information.
NGI-1 inhibition of glycosylation looks to be very weak in Fig. 5B and Fig. S14B.
Our response: It is important to note that the NGI-1 inhibition assay used a suboptimal NGI-1 concentration to prevent full suppression of MHV infection, which we have found previously. We have added this justification in the Methods section and associated figure legend (Fig. S14A).
“The 5 uM NGI-1 dosage was chosen as it resulted in partial inhibition of glycosylation while not completely blocking MHV infection.”
“This dosage and timing were chosen to partially inhibit the OST complex without fully ablating viral infection, as NGI-1 has been shown previously to be a potent positive-sense RNA virus inhibitor(Puschnik et al., 2017) (Fig. S14)”
Summary model figure at the end would help to communicate the conclusions.
Our response: Thank you for this suggestion. We agree and have added a summary model figure at the end as suggested.
