Chikungunya virus glycoproteins transform macrophages into productive viral dissemination vessels

  1. Zhenlan Yao
  2. Sangeetha Ramachandran
  3. Serina Huang
  4. Yasaman Jami-Alahmadi
  5. James A. Wohlschlegel
  6. Melody M.H. Li

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Abstract

Despite their role as innate sentinels, macrophages are cellular reservoirs for chikungunya virus (CHIKV), a highly pathogenic arthropod-borne alphavirus that has caused unprecedented epidemics worldwide. Here, we took interdisciplinary approaches to elucidate the CHIKV determinants that subvert macrophages into virion dissemination vessels. Through comparative infection using chimeric alphaviruses and evolutionary selection analyses, we discovered for the first time that CHIKV glycoproteins E2 and E1 coordinate efficient virion production in macrophages with the domains involved under positive selection. We performed proteomics on CHIKV-infected macrophages to identify cellular proteins interacting with the precursor and/or mature forms of viral glycoproteins. We uncovered two E1-binding proteins, signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 (eIF3k), with novel inhibitory activities against CHIKV production. These results highlight how CHIKV E2 and E1 have been evolutionarily selected for viral dissemination likely through counteracting host restriction factors, making them attractive targets for therapeutic intervention.

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    Reply to the reviewers

    Reviewer 1 major comments:

    The authors show one configuration of the E1-E2 heterodimer in Figure 4d. As shown, the E1 protein is exterior to the E2 protein and would suggest E1 is on the surface on the spike complex and virus surface. However, another configuration of the glycoproteins has E2 on the exterior of E1 and also on the exterior of the virus. The latter conformation is what has been observed in cryoEM studies of alphaviruses. The first configuration represents the E1-E2 between the three heterodimers which are important for spike assembly. The reason the orientation of the E2-E1 dimer is important is the authors speculate on the importance of the 6 CHIK residues not found in ONNV based on the structure, but the structural interpretation is, in my opinion, not correct.

    We thank reviewer 1 for pointing out the correct E2-E1 heterodimer configuration. To address this, we corrected the position of E2 and E1 in Figure 4 based on previous cryoEM study1, keeping E2 always on the exterior in the E2-E1 heterodimer. We also replaced the Indian Ocean Lineage (IOL) E2-E1 structure1 in the original Figure 4 with the CHIKV 181/clone 25 structure which was recently analyzed by Katherine Basore et al.2. In a single E2-E1 heterodimer, all six unique CHIKV positive selection sites are located on the outside of the structure after correcting the configuration. In addition, we investigated two of the unique CHIKV positively selected sites that are important for virion production, E2-V135 (V460 in the original manuscript version) and E1-V220 (V1029 in the original manuscript version), in trimerized structure of E2-E1 heterodimers. We found that the E2-V135 and E1-V220 residues in one heterodimer are facing E2 of the neighboring heterodimer on either side. Interestingly, while V135 is embedded between the E2 proteins of two different heterodimers, E1-V220 is partially embedded by E1 and the neighboring E2 and partially exposed to the outside. This suggests that even though both E2-V135 and E1-V220 might be crucial for CHIKV E2-E1 trimerization, E1-V220 provides an additional docking site for host factor interactions. We thank review 1 again for this important comment leading to these new findings. We have updated Figure 4F-4G and the corresponding result section (lines 201-209) in this partially revised manuscript.

    1. Validation of E1 interaction with SPSC3 and eIF3k needs to be stronger. Some concerns/questions are listed below. A myc tag was inserted between E3 and E2. How efficiently does furin cleave E3 from E2 in this virus and how are viral titers of the myc-tagged virus compared to the non-tagged virus? I ask because is the IP looking at what is being pulled down by E2 or E3-myc-E2 that could be part of the spike polyprotein? The authors found E2 interacts with E3, E1 and a list of other host proteins. These results suggest several interactions including E2-host factor, E2-E1, E2-E3, E2-E1-host factor, E2-E3-E1, E2-E3-host factor. In figure 6d, and the subsequent conclusions, the authors suggest E1 is interacting with the host factor and do not see E2 alone and very low amounts of E3-E2-6K-E1. based on how the IP was performed I am not sure how an interaction between E1 and SPCS3 alone, without E2, would be detected. I would also like to see a reciprocal pull down using E1 and also E2 to see if these host factors are pulled down.

    We thank the reviewer for these concerns. Given the low viral protein expression in macrophages (Figure 1A), we need an efficient system to enrich for large amounts of CHIKV glycoproteins for identifying host interactors through mass spectrometry. Adding tag/reporter proteins, such as mCherry, between E3 and E2 have been used to label alphavirus glycoproteins in previous study2, which is why we chose to use this myc tag labeling strategy coupled with myc Ab-conjugated agarose beads for AP-MS. However, like reviewer 1 speculated, inserting myc tag between E3 and E2 does attenuate CHIKV infectivity according to the reduced supernatant viral titers of 293T cells transfected with CHIKV/myc-E2 genomic RNA in comparison to those of cells transfected with unmodified CHIKV vaccine strain 181/clone 25 genomic RNA (shown in revision plan). Despite the attenuation, CHIKV/myc-E2 harvested from transfected 293T cells still reaches a titer over 108 pfu/ml, which allowed us to identify interactors by AP-MS.

    We further analyzed the cleavage efficiency of glycoproteins by comparing the expression levels of E3-E2-6K -E1, E3-E2 (p62), E2, and E3 in 293T cells transfected with unmodified CHIKV or CHIKV/myc-E2 genomic RNA (result shown in revision plan). We didn’t detect any uncleaved forms of glycoproteins in cells transfected with either unmodified CHIKV or CHIKV/myc-E2 RNA when we probed with E2 antibody. However, probing with E3 antibody prior to longer exposure of the immunoblot showed higher E3-E2-6k-E1 and E3-E2 (p62) levels in cells transfected with CHIKV/myc-E2 RNA, suggesting that both mature E2 and E2-containing precursor polyproteins are available to be pulled down. Overall, the expression levels of mature E2 detected by E2 antibody are similar.

    We thank reviewer 1 for providing a thorough dissection of all the possible interactions between the identified host factors and cleaved/uncleaved glycoproteins. This is a very interesting question. As reviewer 1 mentioned that E1 usually appears with E2 or E3-E2 in heterodimer forms, we were also surprised to find that E2 does not interact with either of the two host factors. To address this, we plan to conjugate E2 and E1 to protein A/G beads, respectively, for a reciprocal pulldown to validate CHIKV glycoprotein interactions with SPCS3 and eIF3k. Results from this experiment will be included in the fully revised manuscript.

    1. If CHIK E1 is interacting with the host factors and that is antagonizing the antiviral response of SPSC3 (as one example), then what do pull downs using ONNV structural proteins look like? One would expect reduced interactions because the different amino acid causes a different E2-E1 dimer or attenuates the E1-host factor binding site.

    We thank Reviewer 1 for this insightful suggestion. We agree that it would be informative to examine the interactions between ONNV glycoproteins and identified host factors (SPCS3 and eIF3k). Unfortunately, there is no commercial ONNV glycoprotein antibody available making this experiment unfeasible. Interestingly, we did observe reduced interactions between the host factors SPCS3 and eIF3k and the CHIKV E1-V220I mutant (V1029I in original manuscript version) where the positively selected site in E1 was mutated to the homologous ONNV residue (please refer to our response to Reviewer 3’s major comment #1). This result suggests that the ONNV glycoproteins likely have an attenuated E1-host factor binding site as the reviewer speculated.We have included this as Figure 7A in partially revised manuscript.

    1. E1 and E2 are thought to interact during polyprotein translation and the initial dimer forms in the ER. If E1 is interacting with SPSC3 in the ER, is E2 also present? Or is a population of E1 not interacting with E2 in order to inhibit SPSC3? I would love a model of how the authors see all these factors coming together for this new role of E1.

    We thank Reviewer 1 for proposing this interesting hypothesis. Given the unexpected absence of E2 in our validation of host factor-E1 pulldown, we speculate that a group of free E1 proteins with distinct function is interfering with host factors in the ER, which is a model worth further investigation and discussion. A great example of this is the alphavirus nonstructural protein 3 (nsP3) that plays essential roles in RNA replication, although depending on the alphavirus not all of the nsP3 in the cell colocalizes with dsRNA, suggesting there is a separate distinct pool of nsP3 outside of active viral replication complex that interacts with host factors in these observed larger cytoplasmic aggregates3. To address this, we plan to use laser confocal microscopy to observe the interactions between host factors (SPCS3, eIF3k), and CHIKV E2 and E1. We will include this result as well as our proposed model in the fully revised manuscript.

    Reviewer 1 minor comments:

    1. In Figure 1c, (-) RNA is shown but in the rest of the figures (+) RNA is shown. Show both or select one. I do find it interesting the (-) RNA levels are similar over time, even at 4 hours post transfection (early time). Related to this, ONNV has higher levels of (-) RNA but what is known about structural protein levels in ONNV and CHIK in macrophages? Are there comparable levels of CP and GP being produced?

    We thank Reviewer 1 for this comment. The (-) RNA is synthesized before the synthesis of subgenomic mRNA and therefore can reflect more accurately early viral replication and nonstructural protein functions. This is the reason why we consider the (-) RNA levels evaluated by specific nsP1 TaqMan probes to be more appropriate for determining early stage differences between ONNV and CHIKV replication in Figure 1 as the goal of that figure is to define the steps in CHIKV life cycle that are more efficient than those of ONNV in THP-1 derived macrophages. On the other hand, the (+) RNA evaluated by E1 primers that we used in the later figures monitors viral RNA synthesis over time in the reflection of genomic (+) RNA and subgenomic mRNA transcribed from (-) RNA templates. Similar levels of (+) RNA and contrasting virion titers really point the difference to the later stages of subgenomic mRNA translation, viral glycoprotein secretion, and assembly.

    We have generated ONNV/myc-E2 reporter virus and assessed viral glycoprotein expression through flow cytometry using a FITC -conjugated anti-myc antibody in the THP-1 derived macrophages transfected with CHIKV/myc-E2 and ONNV/myc-E2 (shown in revision plan). The results show that the expression of ONNV glycoproteins is more inhibited than that of CHIKV glycoproteins, though both of their expression levels in macrophages seem to be suppressed. Since there is no commercial ONNV antibody available, we were unable to compare capsid expression levels between the two viruses. Overall, differences in the myc-tagged glycoprotein expression levels of the two viruses reveals ONNV defect in either structural protein translation or glycoprotein maturation .

    1. Figure 2e and figure 3 have ONNV has the first bar followed by CHIK. In figure 1 and 2b, CHIK is first and then ONNV. helps the reader to have the controls in the same order.

    We thank Reviewer 1 for this suggestion. We have changed the order of ONNV and CHIKV bars in figure 2E and figure3 so the CHIKV bar consistently comes first in all the figures.

    1. Line 143-145 the authors discuss that when ONNV is the backbone and CHIK proteins are inserted the infection is more attenuated because of the E2 and E1 are from CHIK and ONNV, not the same virus (could also be E2-CP interactions are disrupted). However the chimeras made with the CHIK backbone (in Figure 2) have a mismatch between E2 and E1 as well.

    We thank Reviewer 1 for this informative comment. We agree that the incompatible E2-E1 heterodimer formation may not be the only reason that causes attenuation of ONNV/CHIKV E1 and ONNV/CHIKV E2. There may be multiple factors contributing to the fitness of the chimeras, which requires more in-depth mechanistic investigations and is out of the scope of this study. We have now removed the explanation “potentially due to incompatible heterodimer formation between ONNV E2 and CHIKV E1” in line 144.

    1. When discussing the residues that were found in the FEL and MEME analysis, the authors start the amino acid numbering from CP and continue along the polyprotein. Usually when discussing amino acids in the structural proteins, each protein starts at amino acid 1. So V460 would be E2-V135. It would also be useful to know what the residues in ONNV were at these positions to see if amino acids changed in charge, size, bond forming potential, etc. Showing these residues in the E2-E1 conformation found in the virion would also allow one to find adjacent residues that could explain differences in spike assembly and potentially where/how E1 is binding to a host protein.

    We thank Reviewer 1 for this comment. We revised the amino acid numbers in the manuscript to start from the beginning of each structural protein. To look more into these residues in ONNV, we aligned CHIKV and ONNV from different lineages and compared the 6 positively selected sites (refer to our response to Reviewer 1’s minor comment #5). We found that E2-135 and E1-220 which are essential for CHIKV production are valines in all the aligned CHIKV strains. For the aligned ONNV strains, E2-135 are all leucines and E1-220 are all isoleucines. While valine, leucine and isoleucine are all amino acids with hydrophobic side chains, valine has the shortest side chain. The length of the side chains may lead to different hydrophobic properties that affect protein folding, which warrants further structural analysis.

    1. How effective is a non-attenuated CHIK strain in infecting macrophages? Could you make a SINV-La Reunion chimeric virus (which is BSL2) to see if a higher percentage of macrophages are infected and is this potentially contributing to the increased pathogenesis of La Reunion? Also how different is 181/25 with a pathogenic strain in the E2 and E1 residues? and compared to ONNV?

    We thank Reviewer 1 for this question, which is also raised by Reviewer 2. In order to address this question, we plan to use the virulent CHIKV La Reunion strain to study the infection of THP-1 derived macrophages with non-attenuated CHIKV in BSL-3. We are getting trained in the BSL-3 facility and will soon be certified.

    We thank Reviewer 1 for this insightful suggestion on investigating the conservation of these positively selected sites in different strains. We have aligned the sequences of ONNV and CHIKV strains from different lineages, including CHIKV vaccine strain 181/clone 25 and Thai strain AF15561 (the parental strain of CHIKV 181/clone 25) (alignment shown in revision plan). We found that the two positively selected sites with negative effects on virion production, E2-135 and E1-220 (sites 460 and 1029 in original manuscript version), are very conserved in either CHIKV or ONNV strains. CHIKV E2-135 is always valine (V) regardless of the lineages, while ONNV E2-135 is always leucine (L). CHIKV E1-220 is always V, while ONNV E1-220 is always isoleucine (I).

    We also analyzed the amino acid heterogeneity of E2-135 and E1-220 in 397 CHIKV patient sequences from NCBI Virus database. Most of the amino acids at these 2 sites are V. The counts of each amino acid at E2-135 and E1-220 is summarized in table below. This result suggests that valine residues at E2-135 and E1-220 are crucial for CHIKV fitness and strongly selected during viral evolution. The sequence alignment and table will be included and discussed in the fully revised manuscript .

    E2-135

    E1-220

    Valine (V)

    394

    392

    Alanine (A)

    1

    3

    Methionine (M)

    1

    0

    Glutamic acid (E)

    0

    1

    Glycine (G)

    1

    0

    Isoleucine (I)

    0

    1

    1. When describing the last results section, "CHIKV E1 binding proteins exhibit potent anit-CHIV activities" the authors use macrophages. In the rest of the text they consistently use THP-1 macrophages or human primary monocyte derived macrophages. The details of the cell type are extremely useful to the reader and having those in the last results section would be great.

    We thank Reviewer 1 for pointing out the importance of cell type clarification in the last results section. We now consistently use “THP-1 derived macrophages” instead of “macrophages” in this section.

    1. The paper is well-written. There is a slight disconnect as the authors go from discussing results in Figure 4 to Figure 5.

    We thank Reviewer 1 for the comment regarding the disconnection of the last two figures in this paper which is also shared by the other reviewers. We have taken 3 approaches to address this comment: 1) We performed a pulldown of the host factors (SPCS3, eIF3k) identified in Figure 5 with CHIKV positively selected mutants examined in Figure 4 with deficient virion production. The result is presented in our response to Reviewer 3’ s major comment #1, suggesting that the positively selected site in E1 is essential for CHIKV glycoprotein interaction with host factors. 2) To complement our first experiment, we will also determine structural protein expression and processing of parental and E1 mutant CHIKV in eIF3k CRISPR knockout 293T cells. 3) Finally, we plan to perform CORUM analysis to identify high confidence functional protein complexes using our 14 hits found in both mass spec experiments, which will provide mechanistic insights into how these identified cellular complexes and processes might modulate CHIKV infection.

    Reviewer 2’s major comments

    The authors elegantly demonstrate that CHIKV structural proteins confer an advantage over ONNV structural proteins in a step in the replication cycle downstream of virus RNA synthesis, possibly virion assembly. This point would be strengthened determining the particle-to-PFU ratio of the parental viruses and the chimeras . Presumably, the ratio would increase in the chimeras containing CHIKV structural proteins.

    We thank Reviewer 2 for this comment. We agree that determining particle-to-PFU ratios of parental and chimeric viruses will strengthen this study. To obtain the particle-to-PFU ratio, we infected THP-1 derived macrophages with CHIKV, ONNV and chimeras containing CHIKV glycoproteins (Chimera I, and ONNV/CHIKV E2+E1) for 24 h. To quantify the secreted viral particles, we extracted viral RNA in the supernatant and detected (+) viral RNA through TaqMan assay with specific nsp1 probes. The released infectious virions were evaluated through plaque assay. The particle-to-PFU ratios are summarized in the table below. The results show that ONNV has the highest particle-to-PFU ratio (41398), suggesting defective ONNV genome encapsidated in particles leading to defective virion production. On the other hand, the particle-to-PFU ratio of CHIKV (747) is 55-fold lower than that of ONNV. Replacing E3-E2-6K-E1 of ONNV with CHIKV homologous proteins reduces the particle-to-PFU ratio by 8 fold to 4875. Replacing E2 and E1 of ONNV with the ones from CHIKV (ONNV/CHIKV E2+E1) reduces the particle-to-pfu ratio by 20 fold to 2017, suggesting that CHIKV glycoproteins enhance the infectivity of viral progenies produced by THP-1 derived macrophages. We have included the results in Figure 3D-3E in our partially revised manuscript and described in lines 149-158.

    1. Additionally, the authors should consider performing virion assembly blocking assays with a small molecule inhibitor to determine if this abrogates the virus production advantage of CHIKV structural proteins within the ONNV backbone.

    We thank Reviewer 2 for this insightful comment. As the secretory pathway is commonly important for alphavirus glycoprotein maturation and assembly, it will be informative to interrogate CHIKV glycoprotein trafficking and assembly through this pathway using specific inhibitors, such as dihydropyridine FLI-06 and golgicide A . Golgicide A is a reversible inhibitor of the cis-Golgi GBF1, which leads to rapid disassembly of the Golgi and trans-Golgi network (TGN)4. FLI-06 is a new inhibitor that interferes with cargo recruitment to ER-exit sites and disrupts Golgi without depolymerizing microtubules or interfering GBF15. We pretreated THP-1 derived macrophages with 10 uM FLI-06 or golgicide A for 30 mins prior to infection with CHIKV, ONNV, Chimera I, or ONNV/ CHIKV E2+E1. After 1 hour of virus adsorption in PBS with 1% FBS in the absence of the inhibitors, the cells were treated with the inhibitors at the same concentration (10uM) in complete medium for 24 h. The plaque assay result shows that all the viruses are sensitive to secretory pathway inhibition, however, the production of viruses containing CHIKV glycoproteins is significantly more attenuated by FLI-06 and golgicide A. This suggests that CHIKV glycoproteins-mediated trafficking and assembly is more heavily dependent on the host secretory pathway . We will include this result in the fully revised manuscript.

    1. Finally, the authors should perform competition experiments with the chimeric viruses and ONNV in macrophages to determine if the chimeras can outcompete the parental ONNV strain. Based on their data, the chimeric viruses should outcompete.

    We thank Reviewer 2 for this inspiring suggestion. The competition experiment is an innovative and informative way to evaluate whether CHIKV glycoproteins confer a selective advantage on virion production in THP-1 derived macrophages. We plan to infect THP-1 derived macrophages with ONNV and ONNV/CHIKV E2+E1 and detect the viral glycoproteins secreted in the supernatant by western blot, although there is a possibility that this experiment might not work due to superinfection exclusion. Given that there is no commercial antibody of ONNV available, we need to use tagged viruses for this competition experiment. We constructed ONNV/CHIKV myc-E2+E1 that has a myc tag at the N-terminus of CHIKV E2, and ONNV/HA-E2 that has a HA tag at the N-terminus of ONNV E2. Our first attempt at concentrating the viral progenies released by THP-1 derived macrophages infected with the two tagged viruses has not been successful. We performed sucrose gradient ultracentrifugation of the supernatant viral particles but the myc and HA tags were not detected in the expected sucrose layer. Next, we plan to use myc-Ab and HA-Ab conjugated beads to pull down the supernatant viral particles to detect the ratio of ONNV/CHIKV myc-E2+E1 and ONNV/HA-E2 secreted by THP-1 derived macrophages. This will determine whether ONNV containing CHIKV glycoproteins can outcompete ONNV in co-infected cells due to increased viral fitness.

    1. The authors use both primary macrophages and macrophage cell lines as their in vitro model system and make one of their major points (listed in the title) that the determinants they identified in the CHIKV structural proteins convert macrophages into dissemination vessels; however, they do not show: 1) an in vivo model that the CHIKV-ONNV chimeras disseminate more efficiently than the parental ONNV; and 2) that these chimeras generate virus more efficiently specifically in macrophages. It would be useful to show that ONNV and CHIKV have equivalent virion production in other cell lines and that the advantage conferred by CHIKV structural proteins in the ONNV backbone is specific to macrophages. The authors should also change their title to reflect that dissemination is not directly being addressed in their study; the implications of their in vitro experimentation in a mammalian host would be more appropriate for the discussion.

    We acknowledge the limitations of the study, which include a lack of direct demonstration of in vivo dissemination. To address these concerns, we will include further discussion of our in vitro findings in the context of viral dissemination in mammalian hosts in the fully revised manuscript. We are also testing ONNV, CHIKV, Chimera I and ONNV/CHIKV E2+E1 infections in 293T cells to investigate whether the advantage conferred by CHIKV glycoproteins are macrophage specific.

    We have also updated the title to accurately reflect the significance of this research: “Chikungunya virus glycoprotein targeting of host factors increases viral fitness in human macrophage”.

    Reviewer 2’s optional comments

    1. The authors use CHIKV-ONNV chimeras but it would be interesting to test other chimeras to determine if CHIKV structural proteins confer the same advantage in the backbone of other arthritogenic alphaviruses. The study would also be strengthened by using a pathogenic strain of CHIKV instead of the vaccine strain, as this is significantly attenuated in vivo.

    We thank Reviewer 2 for this suggestion which is also suggested by Reviewer 1 in their minor comment #5. We plan to use virulent CHIKV La reunion strain and carry out infection experiments in BSL-3 to strengthen this study. We are getting trained in the BSL-3 facility and will be certified soon.

    1. In Figure 4, the authors identify residues in the CHIKV structural proteins that appear to be under positive selection in human subjects and generate point mutants in these residues with the corresponding ONNV residues. They find that one mutation, V1029I located in E1, completely abolishes virion production in THP-1 macrophage cell lines. However, in their previous chimeric experiments, they find that neither CHIKV E1 or E2 was sufficient to increase virus production in the ONNV backbone. The authors should address this discrepancy, otherwise they should consider moving the data in their point mutation experiments to a supplementary figure. While worthy of reporting, especially given the patient data, these experiments do not buttress the points made in the previous figures.

    We thank Reviewer 2 for this insightful comment. According to previous studies, E2 and E1 always interact with each other from the step of the formation of single heterodimer in the ER to heterodimer trimerization before viral particle assembly. Although the E1-V220 site (previously called V1029) on the exterior of a single E2-E1 heterodimer appears to not be engaged in the E2-E1 interaction E1-V220 is partially exposed and protruding into the groove formed by E1 and the E2 of neighboring heterodimer, accessible to host factors. As such, mutating CHIKV E1-V220 to the ONNV residue (E1-V220I) may not only disrupt E2-E1 trimerization but also interfere viral glycoprotein interaction with host factors(presented in our response to Reviewer 1’s major comment #1). Similarly, solely swapping E2 or E1 with CHIKV substitute in the ONNV backbone would also affect the interaction between neighboring E2 and E1 in trimerized spike, which may explain why neither ONNV/CHIKV E2 or ONNV/CHIKV E1 rescues virion production in THP-1 derived macrophages . We have included this in the partially revised discussion section __lines __ 296-313.

    1. The authors conclude their manuscript with an assessment of several host proteins, namely SPCS3 and eIF3k, that were identified by mass spectrometry and whose knockdown results in increased virion production. The authors speculate about the role of these proteins but do not provide any mechanistic detail on how they might be playing a role. It is unclear that the putative antiviral role of these proteins involves steps downstream of virus replication, especially given that the authors speculate translation might be affected by eIF3k which, if the case, RNA synthesis should also be expected to be affected.

    We thank Reviewer 2 for this comment. We acknowledge that we have yet a full mechanistic understanding of how SPCS3 and eIF3k impact virion production. We plan to investigate their antiviral roles in our follow-up studies. For our partial revision, we have constructed several single eIF3k knockout (KO) clones of 293T cells. The eIF3k sgRNA we designed targets exon 3 which would eliminate expression of all 3 splice isoforms of eIF3k (KO schematic and sequence verification of CRISPR KO shown in revision plan). Unfortunately, we failed to obtain single clones of 293T cells with SPCS3 complete KO, consistent with a previous study by Rong Zhang et al6 that were unable to recover SPCS3 KO clones likely due to the importance of SPCS3 in cell survival. We infected an eIF3k KO clone (clone 9) with CHIKV vaccine strain 181/clone 25, ONNV SG650, and SINV Toto1101. Interestingly, we found that the antiviral activity of eIF3k is specific to CHIKV as CRISPR KO of eIF3k increases CHIKV production by 2.5 fold but not ONNV or SINV production (shown in revision plan). We have included this in the partially revised manuscript in__ line 272-282 (Figure 7B-7D).__

    We presume that Reviewer 2’s inference of eIF3k’s potential effects on viral RNA synthesis is based on our speculation of its antiviral role in viral translation, which may affect viral nonstructural gene expression. We would like to clarify that eIF3k is not an initiation factor traditionally needed for cap-dependent translation. It is also not clear what translation process (nonstructural polyprotein translation from viral genomic RNA or structural polyprotein translation from viral subgenomic mRNA) involves eIF3k if it indeed affects viral protein expression. Notably, previous SINV studies imply that alphavirus structural polyprotein translation may employ unique mechanisms without the requirement of several crucial initiation factors4,5. It will be interesting to see whether eIF3k participates in viral subgenomic mRNA translation as that would affect viral glycoprotein expression leading to reduced virion production. We have now included additional discussion on eIF3k antiviral mechanisms in the partially revised manuscript in lines 345-353.

    1. Overall, while the initial chimeric virus and domain swap approach is strong, the manuscript would benefit with a more thorough examination of virion assembly steps and a mechanistic link to virion production. Otherwise, the authors should revise the structure of their manuscript by de-emphasizing points about virion assembly and leave room for other mechanistic explanations of their chimeric data that more clearly link the host antiviral factor/E1 binding studies.

    We thank the reviewer for these positive comments and suggestions. We have addressed this by further interrogating the production kinetics of CHIKV, ONNV, and the chimeras containing CHIKV glycoproteins through determining their particle-to-PFU ratios as well as treating infected cells with secretory pathway inhibitors (refer to our responses to Reviewer 2 major comments #1 and #2). We have also included additional discussion on eIF3k antiviral mechanisms specifically on how it may affect other steps of the viral life cycle in the partially revised manuscript in lines 345-353 (refer to our response to Reviewer 2 optional comment #3).

    Reviewer 3’s critique comments

    1. Overall, the manuscript is well written but in its current state it is more like two different stories because the effects of envelope proteins and list of interactors are not brought together in one story. A possible fix to this problem would be inclusion of ONNV and CHIKV containing env mutations that do and do not restore viral release from macrophages into the pulldown/association experiments shown in Figure 6.

    We thank Reviewer 3 for the insightful suggestions to better connect the first (CHIKV determinants) and second (CHIKV glycoprotein interactors) parts of the manuscript. In response to the Reviewer’s comment, we tested the binding of SPCS3 and eIF3k to CHIKV E1 with E1-V220I (V1029I in original manuscript version) mutation (shown in revision plan) which was shown to abrogate virion production in THP-1 derived macrophages in Figure 4E. We transfected plasmids expressing 3XFLAG-tagged SPCS3/eIF3k or empty vector for 24 h followed by transfection with plasmids expressing either the parental CHIKV vaccine strain 181/clone 25 poly-glycoproteins (E3-myc-E2-6K-E1) or poly-glycoproteins with the E1-V220I mutation. Interestingly, we found that mutating CHIKV E1-V220 to the homologous ONNV residue reduces the binding to either SPCS3 or eIF3k. This result strongly suggests that the positively selected E1-V220 is located in the interaction interface between E1 and SPCS3/eIF3k, confirming the genetic conflict between E1 and these host factors to be one of the major drivers of CHIKV evolution observed at site E1-V220. We have included this result in partially revised manuscript in Figure 7A and in lines 265-271.

    1. The other major issue is the lack of protein data for the viral mutants relative to WT ONNV and CHIKV and assessment of viral RNA in the supernatants to determine whether the block is release or an earlier event since viral RNA levels in the cell seems to be the same or at least normalized.

    We thank Reviewer 3 for pointing out the insufficient clarification of the block leading to defective CHIKV mutant virion production. We previously detected E2 expression from 293T cells transfected with poly-glycoproteins (E3-myc-E2-6K-E1) containing E2-V135L (V460L in original manuscript version), E2-A164T (A489T in original manuscript version), E2-A246S (A571S in original manuscript version) and E1-V220I (V1029I in original manuscript version). We found that only E2-V135L mutation can lead to unexpected E2 cleavage (shown in revision plan) as we mentioned but not shown in the original manuscript. This explains why E2-V135L mutation attenuates infectious CHIKV production.

    The E2 expression of E1-V220I appears to be not affected in 293T cells transfected with poly-glycoproteins with E1-V220I (shown in revision plan ). In addition, the E1-host factor binding result in our response to Reviewer 3’s major comment #1 showed that E1 with the positively selected site mutation V220I can also be successfully expressed in 293T cells after transfection with poly-glycoprotein. Based on these current data, E1-V220I mutation likely abrogates virion production without affecting glycoprotein expression.

    Our previous result of the ONNV particle-to-PFU ratio reveals that ONNV RNA is released but encapsidated in defective particles causing its attenuation in infected macrophages. Thus, even though the glycoproteins of E1-V220I can be expressed, the diminished virion production of CHIKV E1-V220I can still be ascribed to 1) blocked viral particle release and 2) production of defective particles like ONNV. Given that it is not feasible to obtain particle-to-PFU ratio of E1-V220I mutant which fails to form plaques, Reviewer 3’s suggestion to assess the supernatant viral RNA will be a nice approach to address this question. To further address this concern, we plan to transfect THP-1 derived macrophages with CHIKV E1-V220I mutant RNA to detect the intracellular viral glycoprotein expression and supernatant viral RNA levels through western blot and TaqMan assay, respectively.

    1. Lastly, knockdown experiments indicate an effect of things like OAS3 or other innate immune modulators. There are no controls to demonstrate that these are specific to CHIKV infection or if knockdown would assist growth of ONNV as well.

    We also thank Reviewer 3 for the suggestion to check whether the identified host factors specifically target CHIKV or inhibit the infection of ONNV as well. We previously tried but were facing some issues. Since only a small fraction of macrophages can be infected with CHIKV and even a smaller fraction can be infected with ONNV (Figure 1A), it is hard to elucidate the roles of these identified host factors in ONNV infection by siRNA knockdown. We decided to take a more rigorous approach to investigate the antiviral specificity of identified host factors, especially understudied SPCS3 and eIF3k, to different alphaviruses by generating complete knockout 293T single cell clones. Despite the fact that we did not successfully generate SPCS3 complete KO, we obtained an eIF3k KO single cell clone and infected it with CHIKV, ONNV and SINV (refer to our response to Reviewer 2 optional comment #3). We found that eIF3k only has antiviral activity against CHIKV with almost no effects on ONNV or SINV infection. We have included this in our partially revised manuscript in line 272-282 (Figure 7B-7D).

    Reviewer 3's minor comments:

    Other points to consider:

    1. The title does not fit the manuscript findings and should be modified.

    We thank Reviewer 3 for this important comment, which was also brought up by Reviewer 2. We have now changed our title to “Chikungunya virus glycoprotein targeting of host factors increases viral fitness in human macrophage”, which more accurately reflects the significance of our research.

    1. It is unclear why the authors show results for SINV and RRV in Figure 1. Either these should be removed or the viruses should be carried throughout the experiments described in the Figure. Better yet would be to add additional alphaviruses to this analysis to determine if there are additional viruses that act similarly to CHIKV.

    We apologize for the confusion caused by including SINV and RRV results in Figure 1. We intended to show the superiority of CHIKV in infecting primary monocyte derived macrophages among arthritogenic alphaviruses, which we speculate may provide the molecular basis for macrophage-mediated CHIKV dissemination and disease. We would like to keep the SINV and RRV infection results in Figure 1 to highlight the relative susceptibility of macrophages to CHIKV. To echo the additional alphaviruses tested in Figure 1 and bring the story full circle, we included the result of SINV infection of eIF3k CRISPR KO 293T cells in Figure 7B-7D. These results uncover inhibitory activities of eIF3k that are specific to CHIKV.

    1. Is the data presented in Figure 1A significant?

    We thank Reviewer 3 for this question. We infected both THP-1 derived macrophages and primary monocyte derived macrophages with EGFP-expressing alphaviruses each in duplicates for two independent times. The general low expression of EGFP in all virus-infected groups refrains us from drawing conclusions based on statistically significant differences observed with MFI, hence we chose to show representative scatter plots in the original manuscript. To address Reviewers 3's question, we plotted the infected cell (EGFP+) based on the percentages of the experimental duplicates (shown in revision plan), and found CHIKV infection to be the most significantly different from that of the other alphaviruses in primary monocyte derived macrophage . The numbers above the bar charts are the mean percentages of EGFP+ cells.

    1. The justification for inclusion of Figure 4A is lacking. It is unclear what this panel is supposed to be demonstrating.

    This is an excellent suggestion as the host factors identified by AP-MS not only contain interactors of CHIKV mature E2 but also those of uncleaved E2-containing precursor polyproteins. We modified Figure 4A to reflect all E2/E2-containing poly-glycoproteins present in CHIKV-infected cells (shown in revision plan).

    1. There is little justification for the candidates assessed in

    We understand Reviewer 3’s concern. Due to the nature of mass spectrometry studies which predict protein-protein interactions rather than direct functional validation, we acknowledge that we may miss some host candidates that have anti- or pro-CHIKV activities. Although justification of hit selection from mass spectrometry datasets is more difficult than that from CRISPR KO screen datasets, we set up specific criteria to identify host protein candidates with the greatest potential to functionally interact with CHIKV glycoproteins. Most of the proteins we chose to validate (Figure 6a) were identified in both of our independent AP-MS experiments, which both pass through a P-value threshold of 0.05 and log2 fold change of 0.

    1. Extended data Figure 3 is very difficult to read due to the small font size.

    We apologize for the small font in Extended data Figure 3. We plan to replace Figure EV3 ( Extended data 3 in unrevised version) with a CORUM protein-protein interaction network that centers on the significant hits identified by both AP-MS experiments, but includes hits from either one of the two experiments in these functional protein complexes. The figure will be more concise and centralized, and the font will be bigger.

    1. Just to be clear, the blots shown in Figure 6D are different from those depicted in Extended data Figure 4b, because some of them look very similar.

    We thank Reviewer 3 for this question. In Figure 6D, we expressed CHIKV glycoproteins through transfecting CHIKV genomic RNA into 293T cells, while, in Figure 4B, we expressed CHIKV glycoproteins through transfecting poly-glycoprotein plasmid (pcDNA3.1-E3-myc-E2-6K-E1) into 293T cells, which are complementary approaches to express CHIKV glycoproteins to validate their interactions with identified host factors. We have now added schematics to illustrate the different experimental strategies above the figures in this partially revised manuscript (shown in revision plan).

    References:

    Voss, J. E. et al. Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 468, 709–712 (2010). Jose, J., Tang, J., Taylor, A. B., Baker, T. S. & Kuhn, R. J. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells. Viruses 7, 6182–6199 (2015). Götte, B., Liu, L. & McInerney, G. M. The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last. Viruses 10, 105 (2018). Saenz, J. B. et al. Golgicide A reveals essential roles for GBF1 in Golgi assembly and function. Nat. Chem. Biol. 5, 157–165 (2009). Krämer, A. et al. Small molecules intercept Notch signaling and the early secretory pathway. Nat. Chem. Biol.9, 731–738 (2013). Zhang, R. et al. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature 535, 164–168 (2016).

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Review: In this manuscript the authors generated macrophages derived from the THP-1 cell line or human peripheral blood mononuclear cells stimulated with MCSF and infected them with alphaviruses some containing GFP expression cassettes. In Figure 1, they demonstrate that CHIKV infected these cells more robustly than RRV, SINV or the related ONNV. The authors generated an extensive array of CHIKV/ONNV chimeras to identify the viral proteins that dictate release from infected macrophages and narrowed it down to the envelop proteins E1 and E2. Fine mapping identified a couple of single mutations that affected macrophage infection outcomes. The authors then shifted their approach to identifying env protein interactors using a myc-tag pulldown methods followed by mass spectrometry. The assay identified a number of proteins including those involved in vesicular transport and interferon pathways. siRNA knockdown experiments were performed to identify interactors and many of them were shown to improve virus output.

    Critique: Overall, the manuscript is well written but in its current state it is more like two different stories because the effects of envelop proteins and list of interactors are not brought together in on one story. A possible fix to this problem would be inclusion of ONNV and CHIKV containing env mutations that do and do not restore viral release from macrophages into the pulldown/association experiments shown in Figure 6. The other major issue is the lack of protein data for the viral mutants relative to WT ONNV and CHIKV and assessment of viral RNA in the supernatants to determine whether the block is release or an earlier event since viral RNA levels in the cell seems to be the same or at least normalized. Lastly, knockdown experiments indicate an effect of things like OAS3 or other innate immune modulators. There are no controls to demonstrate that these are specific to CHIKV infection or if knockdown would assist growth of ONNV as well.

    Other points to consider:

    1. The title does not fit the manuscript findings and should be modified.
    2. It is unclear why the authors show results for SINV and RRV in Figure 1. Either these should be removed or the viruses should be carried throughout the experiments described in the Figure. Better yet would be to add additional alphaviruses to this analysis to determine if there are additional viruses that act similarly to CHIKV.
    3. Is the data presented in Figure 1A significant?
    4. The justification for inclusion of Figure 4A is lacking. It is unclear what this panel is supposed to be demonstrating.
    5. There is little justification for the candiates assessed in
    6. Extended data Figure 3 is very difficult to read due to the small font size.
    7. Just to be clear, the blots shown in Figure 6D are different from those depicted in Extended data Figure 4b, because some of them look very similar.

    Significance

    The study provides a fresh look at Alphavirus replication in macrophages. There are a number of issues that should be worked out that would enhance impact and interpretation of this study.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: The authors utilize: 1) chimeric arthritogenic alphaviruses; evolution selection analyses with virus sequences isolated from human patients; and 3) mass spectrometry and proteomics to interrogate determinants of chikungunya virus (CHIKV) permissiveness in primary human macrophages and the human macrophage cell line, THP-1. The authors find that the vaccine strain, CHIKV 181/clone 25 replicates the most efficiently in primary monocyte-derived macrophages compared to other arthritogenic alphaviruses. Using o'nyong o'nyong (ONNV) as a comparison, the authors generate several chimeric viruses with CHIKV structural proteins and ONNV non-structural proteins (and vice versa) and perform a series of E1 and E2 domain swap experiments. They determine that both CHIKV structural proteins, E2 and E1, are necessary to confer efficient virus production over ONNV in the absence of a difference in viral RNA production. The authors also identify a specific residue in E1 that appears to be important for efficient virus production in THP-1 macrophage cell lines. Finally, using mass spectrometry, the authors identify two host proteins, SPCS3 and eIF3k, that bind to CHIKV E1 structural protein and appear to act as antiviral host factors.

    Major comments: The authors elegantly demonstrate that CHIKV structural proteins confer an advantage over ONNV structural proteins in a step in the replication cycle downstream of virus RNA synthesis, possibly virion assembly. This point would be strengthened determining the particle-to-PFU ratio of the parental viruses and the chimeras. Presumably, the ratio would increase in the chimeras containing CHIKV structural proteins. Additionally, the authors should consider performing virion assembly blocking assays with a small molecule inhibitor to determine if this abrogates the virus production advantage of CHIKV structural proteins within the ONNV backbone. Finally, the authors should perform competition experiments with the chimeric viruses and ONNV in macrophages to determine if the chimeras can outcompete the parental ONNV strain. Based on their data, the chimeric viruses should outcompete. These experiments would likely take 3-4 weeks to complete.

    The authors use both primary macrophages and macrophage cell lines as their in vitro model system and make one of their major points (listed in the title) that the determinants they identified in the CHIKV structural proteins convert macrophages into dissemination vessels; however, they do not show: 1) an in vivo model that the CHIKV-ONNV chimeras disseminate more efficiently than the parental ONNV; and 2) that these chimeras generate virus more efficiently specifically in macrophages. It would be useful to show that ONNV and CHIKV have equivalent virion production in other cell lines and that the advantage conferred by CHIKV structural proteins in the ONNV backbone is specific to macrophages. The authors should also change their title to reflect that dissemination is not directly being addressed in their study; the implications of their in vitro experimentation in a mammalian host would be more appropriate for the discussion.

    OPTIONAL: The authors use CHIKV-ONNV chimeras but it would be interesting to test other chimeras to determine if CHIKV structural proteins confer the same advantage in the backbone of other arthritogenic alphaviruses. The study would also be strengthened by using a pathogenic strain of CHIKV instead of the vaccine strain, as this is significantly attenuated in vivo. In Figure 4, the authors identify residues in the CHIKV structural proteins that appear to be under positive selection in human subjects and generate point mutants in these residues with the corresponding ONNV residues. They find that one mutation, V1029I located in E1, completely abolishes virion production in THP-1 macrophage cell lines. However, in their previous chimeric experiments, they find that neither CHIKV E1 or E2 was sufficient to increase virus production in the ONNV backbone. The authors should address this discrepancy, otherwise they should consider moving the data in their point mutation experiments to a supplementary figure. While worthy of reporting, especially given the patient data, these experiments do not buttress the points made in the previous figures.

    The authors conclude their manuscript with an assessment of several host proteins, namely SPCS3 and eIF3k, that were identified by mass spectrometry and whose knockdown results in increased virion production. The authors speculate about the role of these proteins but do not provide any mechanistic detail on how they might be playing a role. It is unclear that the putative antiviral role of these proteins involves steps downstream of virus replication, especially given that the authors speculate translation might be affected by eIF3k which, if the case, RNA synthesis should also be expected to be affected.

    Overall, while the initial chimeric virus and domain swap approach is strong, the manuscript would benefit with a more thorough examination of virion assembly steps and a mechanistic link to virion production. Otherwise, the authors should revise the structure of their manuscript by de-emphasizing points about virion assembly and leave room for other mechanistic explanations of their chimeric data that more clearly link the host antiviral factor/E1 binding studies.

    Minor comments: In Figure 3e, the line under "with CHIKV E1" should be moved over to include the E2-II+E1 virus.

    Figure 5a, 5b, and 6a should be replaced with higher resolution images.

    Significance

    Strengths of the study include the initial chimeric virus and domain swap approach to determine factors that allow for the productive replication of chikungunya virus in macrophages compared to other arthritogenic alphaviruses. This approach yielded useful insights and could be adapted to other viruses. The study is limited, however, by the lack of mechanistic detail linking the antiviral host factors identified which bind to the E1 structural protein, and the advantage conferred by CHIKV structural proteins in the ONNV backbone. The study would be greatly improved by structural studies of the chimeric viruses that directly demonstrate more efficient virion production and that knockdown of the identified factors specifically affects virion production. This point could be addressed either through additional experimentation or tempering of the authors' conclusions about the mechanism by which CHIKV structural proteins provide an advantage over those of ONNV.

    The study advances knowledge in the field on what might advantage different pathogenic alphaviruses and explain differences in disease pathology. Additionally, the authors devise a simple and clever strategy that could be applied across different alphaviruses and would be useful to test in vivo in future studies. This study would be useful to a virology-specific audience.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this work Yao et al. show CHIK is able to infect macrophages in contrast to other arthritogenic alphaviruses RRV, ONNV, and SINV. They use a series to chimeric viruses made with ONNV, the closest species to CHIK, and determine the E2-E1 proteins are important viral determinants which allow CHIK to replicate in machophages compared to ONNV. By comparing 397 CHIK sequences from infected patients, they identified 14 residues under pervasive and positive selection. Of these, 3 residues in E2 and 3 residues in E1 (amino acids) were different between CHIK and ONNV suggesting these residues contributed to the difference in macrophage tropism of CHIK compared to ONNV. The authors go on to determine what host factors the CHIK E2 protein is interacting with to presumably connect the viral and host determinants for CHIK infection in macrophages.

    Major concerns:

    1. The authors show one configuration of the E1-E2 heterodimer in Figure 4d. As shown, the E1 protein is exterior to the E2 protein and would suggest E1 is on the surface on the spike complex and virus surface. However, another configuration of the glycoproteins has E2 on the exterior of E1 and also on the exterior of the virus. The latter conformation is what has been observed in cryoEM studies of alphaviruses. The first configuation represents the E1-E2 between the three heterodimers which are important for spike assembly. The reason the orientation of the E2-E1 dimer is important is the authors speculate on the importance of the 6 CHIK residues not found in ONNV based on the structure, but the structural interpretation is, in my opinion, not correct.
    2. Validation of E1 interaction with SPSC3 and eIF3k needs to be stronger. Some concerns/questions are listed below. A myc tag was inserted between E3 and E2. How efficeintly does furin cleave E3 from E2 in this virus and how are viral titers of the myc-tagged virus compared to the non-tagged virus? I ask because is the IP looking at what is being pulled down by E2 or E3-myc-E2 that could be part of the spike polyprotein? The authors found E2 interacts with E3, E1 and a list of other host proteins. These results suggest several interactions including E2-host factor, E2-E1, E2-E3, E2-E1-host factor, E2-E3-E1, E2-E3-host factor. In figure 6d, and the subsequent conclusions, the authors suggest E1 is interacting with the host facor and do not see E2 alone and very low amounts of E3-E2-6K-E1. based on how the IP was performed I am not sure how an interaction between E1 and SPCS3 alone, without E2, would be detected. I would also like to see a reciprocal pull down using E1 and also E2 to see if these host factors are pulled down.
    3. If CHIK E1 is interacting with the host factors and that is antagonizing the antiviral response of SPSC3 (as one example), then what do pull downs using ONNV structural proteins look like? One would expect reduced interactions because the different amino acid causes a different E2-E1 dimer or attenuates the E1-host factor binding site.
    4. E1 and E2 are thought to interact during polyprotein translation and the initial dimer forms in the ER. If E1 is interacting wht SPSC3 in the ER, is E2 also present? Or is a population of E1 not interacting with E2 in order to inhibit SPSC3? I would love a model of how the authors see all these factors coming together for this new role of E1.

    Minor concerns:

    1. In Figure 1c, (-) RNA is shown but in the rest of the figures (+) RNA is shown. Show both or select one. I do find it interesting the (-) RNA levels are similar over time, even at 4 hours post transfection (early time). Related to this, ONNV has higher levels of (-) RNA but what is known about structural protein levels in ONNV and CHIK in macrophages? Are there comparable levels of CP and GP being produced?
    2. Figure 2e and figure 3 have ONNV has the first bar followed by CHIK. In figure 1 and 2b, CHIK is first and then ONNV. helps the reader to have the controls in the same order.
    3. Line 143-145 the authors discuss that when ONNV is the backbone and CHIK proteins are inserted the infection is more attenuated because of the E2 and E1 are from CHIK and ONNV, not the same virus (could also be E2-CP interactions are disrupted). However the chimeras made witht he CHIK backbone (in Figure 2) have a mismatch between E2 and E1 as well.
    4. When discussing the residues that were found in the FEL and MEME analysis, the authors start the amino acid numbering from CP and continue along the polyprotein. Usually when discussing amino acids in the structural proteins, each protein starts at amino acid 1. So V460 would be E2-V135. It would also be useful to know what the residues in ONNV were at these positions to see if amino acids changed in charge, size, bond forming potential, etc. Showing these residues in the E2-E1 conformation found in the virion would also allow one to find adjeacent residues that could explain differences in spike assembly and potentially where/how E1 is binding to a host protein.
    5. How effective is a non-attenuated CHIK strain in infecting macrophages? Could you make a SINV-La Reunion chimeric virus (which is BSL2) to see if a higher percentage of macrophages are infected and is this potentially contributing to the increased pathogenesis of La Reunion? Also how different is 181/25 with a pathogenic strain in the E2 and E1 resdiues? and compared to ONNV?
    6. When describing the last results section, "CHIK E1 binding proteins exhibit potent anit-CHIV activities" the authors use macrophages. In the rest of the text they consistently use THP-1 macrophages or human primary monocyte derived macrophages. The details of the cell type are extremely useful to the reader and having those in the last results section would be great.
    7. The paper is well-written. There is a slight disconnect as the authors go from discussing results in Figure 4 to Figure 5.

    Referees cross-commenting

    I agree with R#2 that having some Particle:PFU data would add some data to determine why such differences in titers/infectivity.

    I also see how this m/s could be split into two different m/s. One that focuses on the chimeric viruses and another that identifies the host factors important and goes in more depth with mechanism

    Significance

    Strengths:

    The authors have tackeled an intriguing question: why do some alphaviruses infect macrophages and others do not. They have used a chimeric approached to very systematically identify the viral determinants E2 and E1 as being important in macrophage infection. Using AP-MS they identify host factors that interact with E2 (possibly E2 and E1, see comments above) but if their findings that E1 has a role in attenuating a host antiviral factor, this would be fantastic.

    More and more examples of viral proteins having multiple roles during infection are in the literature. The idea that structural proteins also attenutate host antivirals is a developing field and vastly understudied. By fleshing out the results some more the authors might be onto something ery important in alphavirus virology.

    Limitations:

    The study has it is presented is limited in the validation of host factors and their interacting partners. I have many questions about the methodology, validation, and model from this last section.

  5. Version published to 10.1101/2023.05.29.542714v1 on bioRxiv