Arteiviridae family, is a major pathogen affecting pigs worldwide. The membrane (glyco)proteins GP5 and M form a disulfide-linked dimer, which is a major component of virions. GP5/M are required for virus budding, which occurs at membranes of the exocytic pathway. Both GP5 and M feature a short ectodomain, three transmembrane regions, and a long cytoplasmic tail, which contains three and two conserved cysteines, respectively, in close proximity to the transmembrane span. We report here that GP5 and M of PRRSV-1 and -2 strains are palmitoylated at the cysteines, regardless of whether the proteins are expressed individually or in PRRSV-infected cells. To completely prevent S-acylation, all cysteines in GP5 and M have to be exchanged. If individual cysteines in GP5 or M were substituted, palmitoylation was reduced, and some cysteines proved more important for efficient palmitoylation than others. Neither infectious virus nor genome-containing particles could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicating that acylation is essential for virus growth. Viruses lacking one or two acylation sites in M or GP5 could be rescued but grew to significantly lower titers. GP5 and M lacking acylation sites form dimers and GP5 acquires Endo-H resistant carbohydrates in the Golgi apparatus suggesting that trafficking of the membrane proteins to budding sites is not disturbed. Likewise, GP5 lacking two acylation sites is efficiently incorporated into virus particles and these viruses exhibit no reduction in cell entry. We speculate that multiple fatty acids attached to GP5 and M in the endoplasmic reticulum are required for clustering of GP5/M dimers at Golgi membranes and constitute an essential prerequisite for virus assembly.Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-strand RNA virus in the  Nidovirales, is an important pathogen for pigs. Despite its importance in veterinary medicine, basic structural and functional features of its membrane proteins have not been elucidated. Here, we provide evidence for palmitoylation of the PRRSV major membrane proteins GP5 and M at a cluster of membrane-near cysteines. Fatty acid attachment is required for virus growth, since removal of all acylation sites from either M or GP5 prevents recue of infectious particles. Furthermore, viruses lacking individual acylation sites in M and GP5 grow to significantly lower titers in cell culture. The specific infectivity and cell entry of viruses lacking two acylation sites in Gp5 is, however, not reduced. Likewise, these viruses revealed no effect on dimerization of GP5 with M, its transport to budding sites, and incorporation into virus particles. Since cells transfected with a cDNA expressing non-acylated GP5, or non-acylated M release no virus-like particles into the supernatant we propose that the fatty acids are required for the budding process. They might trigger assembly of GP5/M dimers to form a coat inside the lipid bilayer that induces membrane curvature.Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus in the order  Arteriviridae family of the order Nidovirales, is one of the most relevant viral pathogens in pigs worldwide. PRRSV infection causes abortion and stillbirth in pregnant sows as well as respiratory disease and poor growth performance in piglets  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Jens H. KuhnAssociate EditorPLOS PathogensCarolina LopezSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: This manuscript by Zhang and colleagues explores the functional impact of palmitoylation at key conserved cysteine residues in the PRRSV membrane proteins GP5 and M. The authors show that, indeed, conserved cysteines in GP5 and M are palmitoylated, and that some cysteines were more efficiently palmitoylated than others. Furthermore, viruses lacking all palmitoylated cysteines in GP5 or M could not be rescued, while single/double cysteine mutant viruses tended to grow to lower titers, suggesting the importance of these conserved amino acids for virus particle production. While the data is interesting, and have the potential to improve our understanding PRRSV assembly, I feel that the study falls short of providing convincing mechanistic evidence for why palmitoylation mutant viruses are defective.Reviewer #2: This submission presents a study of the five to six cysteines on PRRSV GP5 and M proteins that are potentially subject to post-translational palmitoylation. The investigators documented that the cysteines are indeed palmitoylated. They then found that C to S substitutions, and therefore, the complete absence of palmitoyl adducts on GP5 and M, was incompatible with PRRSV production. Under-palmitoylation did, however, allow for virus production and the authors exploited this finding. They found that wild-type palmitoylation, relative to under-palmitoylation, allowed for more virus particle output, most likely due to increased virus budding. Notably the under-palmitoylation had little if any effect on virus entry. The roles for palmitoylation in virus budding, therefore, were discussed relatively thoroughly in this report.Overall, the report appears to be valuable. It is important to know how viral protein palmitates operate in virus infections, and this study points sensibly to palmitoylation requirements in virus budding or some other aspect of particle morphogenesis. This reviewer’s opinion is that the report is well written, the experiments executed well, and the solid results generally interpreted properly. There are, however, still some questions about interpretation of findings and evidence for the conclusions made in the report. These questions are put forward in the comments below.Reviewer #3: In the paper ‘Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus (PRRSV) is essential for virus growth’, Zhang et al. demonstrated that the 3 cysteines of the cytoplasmic tail of GP5 and the 2 cysteines of the cytoplasmic tail of M are palmitoylated (upon transfection and infection). If all cysteines of GP5 or M were replaced no virus could be rescued, demonstrating that acylation is essential. Trafficking of GP5 and M to the Golgi apparatus was not disturbed by the absence of acylation. These results were obtained with a technology that was successfully used before by the senior scientist with other viruses. Therefore, the conclusion that the multiple fatty acids attached to GP5 and M are crucial in the virus assembly can be supported. Since this is a lacking piece of the PRRSV replication puzzle, it is quite interesting. I have more reservations with the technology that was used for demonstrating that the palmitoylation is not important for the entry of the virus. GP5/M is important for the infection of receptor (siglecs)-carrying macrophages. This is not the case for Marc cells. As a result, a virus titer of a wild type virus stock in macrophages is tenfold higher than in MARC-145 cells. This explains the big difference between the TCID50 and genome copies, as also demonstrated in the present study. In order to study the role of palmitoylation of GP5 and/or M it is important to use macrophages that express receptors that interact with GP5/M. Furthermore, it is important to note that RT-qPCR does not only detect genomes inside virions but also free genomes. It would have been better to first eliminate the free genomes by benzonase treatment.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be 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: 1. Fig. 1 A: PRRSV-1 strain Lelystad and PRRSV-2 strains VR-2322 and XH-GD serve as prototype references strains. This feels like a very surface level analysis. Given that considerable genetic variation exists between different PRRSV isolates, the authors could strengthen this analysis with a broader panel of sequenced PRRSV isolates. Furthermore, are these conserved cysteines found in other arteriviruses . Is this conservation unique to GP5/M of all arteriviruses? Are there exceptions? This information should be provided up front, rather than waiting until the discussion section.2. RE: “Palmitoylation of M and GP5 was also detected during replication of both viruses in isolated porcine alveolar macrophages, the target cells of PRRSV in pigs (not shown).” PAMs are a more relevant model system than monkey kidney cells (MARC-145), yet that data in PAMs is “not shown.” Either this data should be presented, or this sentence should be removed.3. “…in contrast to the diffuse bands of GP5 in virus infected cells.” In a later figure, GP5 shows up as a double-band under similar reducing conditions. Is this an exposure issue? Are these “diffuse bands” being overexposed in the western blot for 1B compared to 1C? The authors should carefully compare viral infections (which express both GP5/M) to transfections with both GP5/M genes, as well as each gene alone.4. RE: “The results suggest that GP5 and M remain in the ER if expressed alone, similar to what has been reported for…” To make this statement, the authors should repeat the experiment, but with co-transfection of both GP5/M. I think the authors are making too great of a leap here so early in the paper, and this whole paragraph seems out of place. The difference in banding patterns could be due to differences between cell types used, for instance, as N-glycosylation is known to be cell type dependent. However, they then go on to do IF to test their assumptions, but for some reason they switch from MARC-145 (infection) to 293Ts (transfection), and then to making assumptions on GP5/M co-localization with cellular markers in a completely different tissue/species cell line (transfected BHKs) . There should be consistency between the cell types used in these experiments. Further, the authors are measuring co-localization with a golgi marker and making assumptions about ER localization? The authors could easily test this assumption by repeating the experiment, but with an ER marker.5. Fig 2. I appreciate the addition of Flotillin 2 as an internal assay control. This should be “flotillin” not “flotilin" though. It is correct in the text, but incorrect in the figure. Where is the flotillin blot for 2G? Aesthetically speaking, the images should all be cropped similarly. Sometimes the ladder is shown, sometimes it isn’t. Sometimes the 36 kDa band on the gel is shown, sometimes it isn’t. Most blots have a mock, but panel F doesn’t. The consistency in the figure is lacking.6. Fig. 2. When each cysteine is replaced, there is a qualitative difference the amount of GP5 pulled down. This is a really interesting finding, especially given that some cysteines seem to be more important than others in terms of their relative palmitoylation state. I’m really curious why the same wasn’t done for M? Similar experiments for GP5 and M are carried on throughout the paper, but for some reason, not here.7. Fig 2 legend. I’m curious about how the statistics were performed. If each independent experiment was normalized to GP5 wt set at 100%, and then that data was combined, your wt would be 100% with no statistical deviation (as presented in the graph). Then, wouldn’t any statistical comparison you make between your mutants and wt here show a significant difference ? It seems like the combined, non-normalized data, from three experiments  would be the dataset that the statistics should be tested on. Not the normalized data. The authors need to be more specific in how their stats were analyzed in the legend and correct if need be.8. It would be nice to see more consistency throughout the paper. Figure 1 has Lelystad (PRRSV-1) and VR2322 and XH-GD (PRRSV-2). XH-GD is dropped in figure 2, but then viral infections were done with XH-GD through the rest of the paper. For consistency purposes, it would have been nice to also see data for XH-GD in Fig 2 so analogies could be drawn with the same mutants used in transfection/infection experiments.9. Fig. 3 results section:a. In the text, viral cDNA was transfected into 293T cells, but in the legend and figure, it states that BHKs were transfected. So, which cell line was it?b. It is unclear why this transfection step was necessary. If MARC-145 cells “support virus replication,” why then couldn’t all of this been done in those cells? Could the authors briefly provide more experimental details here?c. “…five further attempts to rescue infectious mutant virus also failed.” It is unclear what the parameters were here. Did you do the same thing, five different times, or did you troubleshoot it differently? This sentence alone does not provide much help to the reader.10. Fig. 3: A few points…a. It would be nice to see the nuclear staining in all images. Just showing a black box isn’t informative.b. Is there a reason why GP5 staining, but not M staining, is shown? It would be important to show that mutagenesis did not disrupt the ability of these viruses to express both of these proteins.c. “…we could not detect viral genomes for any of the GP5 and M mutants in the supernatants of infected cells, whereas ~107 genome copies of wild-type virus were present.” I feel that this is the most important point here for Fig 3. There’s no point to show IF infection of MARC-145 cells if no virus particles were produced in the first place. Thus, showing infection via immunofluorescence in MARC-145 cells seems meaningless. Further, for the qPCR data, for all viruses in 3B, are similar genome copy numbers detected in the cell supernatant? This would be an interesting addition to this figure.11. Fig. 3A. For the GP5/M palmitoylation mutants, where no viral genomes were detected in the supernatant: The authors should explore the fate of virions in the producer cells. Do viral genomes still accumulate in the cell? Can you detect intracellular viral particles? Do these proteins get stuck in different organelles, unlike the wild-type proteins which would be expected to traffic normally? More mechanistic underpinnings here would heighten the impact of this work.12. Fig. 4. I appreciate seeing the growth kinetics for the wild-type and mutant viruses. As an important control, are GP5 and M in the mutants being incorporated into virus particles to similar levels as the wild-type proteins?13. Fig. 4. The authors need to address the variability between the replicate experiments. I’m very surprised (and nervous) to see that sometimes there are several logs difference noted between wt and mutant (let’s look at 4B wt vs purple line @ 24h), yet there is no statistical difference between those points. This is a massive amount of experimental variation. The authors cannot conclusively say “each mutant virus grows to lower titers when compared to those of wild-type PRRSV” when all mutant viruses (except 122+131 and 122+138) reach the same endpoint titer that is not statistically different than the wild-type virus. This seems to be a misrepresentation of the data, and that selling point is made multiple times throughout the paper. This is one of the most important figures in the paper, yet the data  doesn’t fit with the authors interpretation.14. Fig. 4 methods: “the authenticity of rescued viruses was confirmed by sequencing, and viruses were further passaged 2-3 times in T175 flask to generate a for virus stock for subsequent experiments.” Was this sequencing performed before, or after virus passaging to generate stocks? How was it performed? Sanger sequencing of a PCR product? Sanger of multiple independently cloned PCR inserts? Next-gen sequencing? Critical details are missing here. I’m curious how stable these mutations were in their virus stocks, and how stringent the authors were in sequencing their stocks is important for data interpretation.15. Fig. 4D. I don’t see the point in just showing the palmitoylation data for M single mutants 99 and 102, in comparison to wild type. If you want to present this level of detail, why then isn’t this being presented for all of the mutant viruses in A and B? I don’t see any rationale for why these two mutants were chosen. Further, the authors state 10 to 1000-fold differences at 48 h, but in the figure, non-significant differences at 12, 24, 72, and 96 hours are noted. This seems like only select information is being highlighted, rather than telling the whole story. The endpoint titer of these mutant viruses are not significantly different than wt, despite the “tendency for lower titers.”16. Fig. 5. How have the authors controlled for equal protein loading in these westerns? No loading controls are shown. For the ratios, I would have expected each band in -ME and +ME to have been internally normalized to a loading control (on the same blot), and then those band intensities could be compared -ME to +ME. Why haven’t the authors similarly probed for M protein in these blots, thereby confirming that the 38 kDa band was a GP5-M heterodimer, rather than speculating? Further, and again to touch on consistency, why is the ladder shown in some but cropped out in others?17. Connecting Fig. 3 and Fig. 5. No infectious virus is produced in the pGP5 SSS, pM SS, or pGP5 SSS + M SS mutants. In Figure 5, the authors suggest that GP5 dimerizes with M . Is the reason for the lack of secreted pGP5 SSS, pM SS, or pGP5 SSS + M SS viruses because these mutant GP5/M proteins do not dimerize together, thus, budding is impeded? If you pull down GP5, can you detect M and vice versa? More mechanistic details here would add additional strength to this work.18. I would have appreciated Fig. 5 more if the authors would have better described now N-glycan addition and Endo-H protection fit with their interpretation that the “same number of molecules have passed through the medial golgi-complex.” I’m not fully understanding how the data fit with their interpretations. Importantly, “…Endo-H resistant bands were also detected if non-acylated GP5-SSS and M-SSS were co-expressed (data not shown).” Why is this data not shown? The authors lack experimental detail for why these mutants failed to make infectious virus. Does this, along with the co-localization experiment , suggest it isn’t due to any trafficking issues?19. Fig. 6. This figure analyzes localization of GP5/S mutants, for those mutants that could not produce infectious virus . How does co-localization of GP5 and M in the golgi allow the authors to conclude that they are indeed dimerized? This is also an overexpression scenario, would is known to cause unintended effects on protein localization.20. Fig. 7. I appreciate that the authors showed incorporation of mutant GP5 proteins in relation to wildtype. How was the blotting normalized for these concentrated viral preparations, especially since the mutant viruses tend to produce lower viral titers than wild-type? Was it by protein input? Careful descriptions are necessary here. Also, why were these particular mutants chosen but not others? In my opinion, this is an important control figure that should be shown for all viruses produced, and it should have been presented several figures back.21. RE: Virus particles with under-palmitoylated GP5 have no significant defect in cell entry. This whole section was very difficult to follow. Two different assays, with two different viruses are being placed into a single table. The table headers aren’t easy to interpret on their own. The infection values are extremely low, and if you are looking at entry, waiting 10 hours to detect N seems very long. I get the point that the authors are trying to make, and it’s an important one, it just doesn’t come off as easily interpretable in the way the data is described and presented. Also, no staining for N is shown , making this experiment difficult to review. If the authors could get a hold of a reporter PRRSV isolate, it would greatly improve their experimental setup here. It could be normalized by genome content, TCID50 could be determined by flow cytometry, and then entry measured by reporter gene expression shortly after infection.22. Overall, I don’t see experimental data to explain why palmitoylation mutant viruses behave differently than wild type. To strengthen this paper, I feel that the authors need to delve deeper into that mechanism. To summarize the major figure conclusions:a. GP5/M are palmitoylated b. Some cysteines are more important for palmitoylation than others c. Removing all cysteines in GP5 or M prevents viral particle release d. Palmitoylation mutants tend to have lower titers e. There is no difference in glycosylation or Endo H protection f. GP5/M mutants co-localize together .g. GP5 mutants incorporate similarly to wt into virions .h. No defects in entry are noted (Table 1).None of this data provides mechanistic insights into why GP5/M need to be palmitoylated, other than reduced palmitoylation of GP5/M has a tendency to impact viral titers.23. The authors speculate that acylation affects the budding process. Could the authors use an ELISA to prove this point? If less viruses are budding out, this should be quantifiable in a protein ELISA, which can then further be compared to viral genome copies in infected cell supernatant.24. Figure 8 is not sufficient as a standalone figure, and I would suggest that these alignments be added to the first figure.Reviewer #2: 1. The report suggests a role for palmitate modifications in virus budding and virus particle membrane curvature. These are reasonable suggestions and the role in budding is somewhat supported by excluding other roles for the palmitoylations. However, there is not much direct evidence for a role for the palmitates in budding. One suggestion is to perform EM of the GP5 2-palm-deficient viruses. Any evidence of misshapen particles, deformed because under-palmitoylation creates aberrant membrane curvatures?2. It is hard to accept one of the main conclusions of the paper regarding palmitates in virus budding. “We concluded that palmitoylation of GP5 does not affect incorporation of the GP5/M dimer into virus particles.” This appears to be based on the single palmitoylated GP5 mutant, lacking two palm cys residues. Any analysis of un-palmitoylated GP5 in virus incorporation could not be performed because palm-negative viruses were not obtained. Therefore, how can it be concluded that palmitoylation of GP5 is not required for incorporation into virus particles?3. The PRRSV cDNAs encoding palm deficient GP5 and M can produce viral proteins but do not release infectious particles. Can the cDNA – transfected cells be complemented with GP5 and M expression vectors, to then produce viruses? Results from such complementation tests would further confirm findings in this study.4. Virus egress via exocytosis is required after budding. Have roles for palmitates on GP5 and M in forming egress-competent viruses been considered, or excluded from consideration for some reason?Reviewer #3: 1° See under SummaryI have reservations with the technology that was used for demonstrating that the palmitoylation is not important for the entry of the virus. GP5/M is important for the infection of receptor (siglecs)-carrying macrophages. This is not the case for Marc cells. As a result, a virus titer of a wild type virus stock in macrophages is tenfold higher than in MARC-145 cells. This explains the big difference between the TCID50 and genome copies, as also demonstrated in the present study. In order to study the role of palmitoylation of GP5 and/or M it is important to use macrophages that express receptors that interact with GP5/M. Furthermore, it is important to note that RT-qPCR does not only detect genomes inside virions but also free genomes. It would have been better to first eliminate the free genomes by benzonase treatment.2° Macrophages are the real target cell. Therefore, some findings in MARC-145 cells should be confirmed in macrophages such as the demonstration that palmitoylation is not required for dimerization of GP5 and M, for transport to the Golgi apparatus and for incorporation into virus particles.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1. The manuscript should be updated with line numbers to help the reviewer address specific lines in the paper.2. Abstract: “no virus could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicated that acylation is essential for virus replication.” I don’t think “replication” is the correct term here. No viral genomes were released for these mutants, but the authors did not show that these mutants had any defect in viral genome replication in the producer cells. Perhaps, viral “egress from the cell” would be more suitable?3. Fig 1 legend: “Note that GP5 also contain cysteines in the middle of the TMR.” These cysteines are immediately adjacent to the cytoplasmic tail. Is “cysteines in the middle of the TMR” correct here?4. “…we exchanged them with serine in GP5-HA and M-His in both the VR-2322 and Lelystad viruses.” And here: “…revealed a remaining palmitoylation level of ~20% in the Lelystad and of only ~10% in the VR-2332 virus.” The way these sentences are worded makes it sound like you made these serine mutations in the viruses themselves, rather than expression vectors. Please double-check and correct this wording throughout.5. Fig. 3 legend and throughout: “Alex-568” should be Alexa-568.6. The manuscript should be thoroughly re-read and corrected for grammar and spelling errors.Reviewer #2: 1. A point about terminology here: The term “replication” often refers to replication of viral genetic material. In this context, then, it is not established that the absence of palmitates eliminates PRRSV “replication”. In fact, replication appears to be demonstrated in the absence of palmitoylated GP5 – M. Palmitates instead are suggested to be needed for particle assembly or budding or perhaps particle egress. The use of the terms “replication” and “replication deficient” might be reconsidered in this manuscript.2. Regarding consensus palmitoylation motifs, are there not predictive algorithms for palmitoylation? (intro stating lack of consensus motifs suggests otherwise).3. Fig. 5; were thiol blockers (NEM or IAA) included during the cell lysis process, to prevent artifactual post-lysis disulfide formations?4. The review process would be more effective if the text included line numbers, so if there is any additional review, a submission with line numbers would help.Reviewer #3: Author summary - last sentence: ‘They might trigger assembly of GP5/M dimers to from…’ change into ‘They might trigger assembly of GP5/M dimers to form…’**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methodsTo enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see  20 Feb 2021AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 15 Mar 2021Dear Dr. Veit,Thank you very much for submitting your manuscript "Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus is essential for virus growth" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the few remaining recommendations by the reviewers (minor revision).Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Jens H. KuhnAssociate EditorPLOS PathogensCarolina LopezSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: In this resubmission, Zhang et. al. have made several important additions to their study, which provides evidence that GP5/M palmitoylation is important for producing infectious PRRS virus particles. I appreciate the detailed point by point responses to the critiques, and providing revisions, clarifications, and additional experiments to the manuscript where appropriate.Reviewer #2: The submission is improved. New results in Fig 5 support a role for palmitoylation in particle assembly or secretion. A revised and clarified presentation of findings in Fig 9 confirm that that viruses containing under-palmitoylated GP5 have similar target-cell infection capacities. The inferences are that palmitoylation operates in virus-producing cells at the level of virus assembly, that some select under-palmitoylation can be tolerated at assembly, and that those under-palmitoylated virus particles that can be assembled and secreted will operate similarly to natural viruses during target cell entry.Reviewer #3: Reviewed version of a paper.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be 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: NoneReviewer #2: This reviewer still has a few questions.1. Fig. 5: Since the vGP5 122 + 138, vM 99 and vM 102 are so prominent in several experiments , why are they not evaluated and included in the Fig. 5 results?2. The new Fig. 5 seems suboptimally placed into the manuscript. After producing mutant genomic cDNAs, the very first test would seem to be the one whose results are shown in Fig. 5. Then, with the results, the next questions could focus on the characteristics of those few recombinant cys-to-ser viruses that were produced and secreted; i.e., their replication kinetics , virion proteins , entry into target cells  and so on.3. This reviewer still has difficult with summary statements. The statement at the end of the abstract refers to “clustering of GP5/M dimers at Golgi membranes” and it is not clear how the findings reported in the body of the abstract support this particular statement. Other summary statements are hard to discern, for example, lines 47-50 of the author summary; “acylation has no effect on dimerization of GP5 with M, its transport to budding sites, and incorporation into virus particles. We therefore propose that the fatty acids are required for the budding process.” It would help to clarify how observation of under-palmitoylated GP5 in virus particles lead one to the stated proposal.Reviewer #3: I agree with the corrections made by the authors.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1. I appreciate the addition of Fig. 1A, and it’s fascinating to see this analysis of sequence variation between isolates. However, it’s difficult to discern which dot in the graph each cysteine is referring to given how scrunched together the data points are. To better distinguish the cysteines, could the authors simply mark those data points in a different color?2. The addition of the PAM experiment  provides important data for a natural host cell target. However, I’m struggling with the M-protein blot. It doesn’t appear as though a -HA sample was loaded given the lack of spacing between the M-specific bands. Can the authors please comment, or perhaps include a new blot here that is more consistent with the others?3. Lines 138-139: For added clarity, please denote which proteins ORF5 and ORF6 encode.4. Line 176: Suggest replacing “confirming” to “consistent with”Reviewer #2: (No Response)Reviewer #3: I agree with the corrections made by the authors.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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Parvoviridae) and an alphapermutotetra-like virus (family Permutotetraviridae) were discovered from transcriptomic data sets obtained from Thaumetopoea pityocampa larvae collected in Portugal. Each of the coding-complete genome sequences of these viruses contain three main open reading frames (ORFs).The coding-complete nucleotide sequences of an iteradensovirus (family  Parvoviridae) and an alphapermutotetra-like virus (family Permutotetraviridae) were discovered from transcriptomic data sets obtained from Thaumetopoea pityocampa larvae collected in Portugal. Each of the coding-complete genome sequences of these viruses contains three main open reading frames (ORFs).The coding-complete genome sequences of an iteradensovirus (family  Bacillus thuringiensis strain kurstaki; this method is now strongly limited in Europe (Pine processionary moth (PPM) caterpillars are a major threat to conifer forests in Mediterranean regions . They lin Europe .Entomopathogenic viruses were considered to control PPM in the 1950s but never developed . Today, Parvoviridae), and an alphapermutotetra-like virus, a single positive-strand RNA virus (family Permutotetraviridae).Here, we describe the complete coding genome sequences of two viruses infecting Lepidoptera found in PPM larvae collected for a population survey in Portugal. Total RNAs were extracted from 2 to 5 third-instar caterpillars with TRIzol reagent, pooled in equimolar amounts. Libraries were constructed using the TruSeq stranded mRNA prep kit (Illumina) with an insert size of 450 bp and sequenced in two lanes of paired-end (PE) 2 × 100-bp reads on the HiSeq 2000 platform  and asseThaumetopoea pityocampa iteradensovirus (TpIDV) has two contigs assembled from 233,972 and 176,824 reads , representing 94% of the total viral reads   is made of one contig  assembled from 20,260 reads , which represent 4.6% of the total viral reads (YP_009361866), 34% amino acid identity with the putative RNA-dependent RNA polymerase (RdRP) from the Hubei leech virus 1 , and 49% amino acid identity with the capsid protein (CP) of the Thosea asigna virus , the type species of the genus Alphapermutotetravirus (http://rna.urmc.rochester.edu/RNAstructureWeb) at the 3′ end might correspond to minor CP(s) and pseudoknots described in this virus family  NCBI database under BioProject accession number PRJNA663237.The GenBank accession numbers are


Myotonic dystrophy (DM) is a rare, inherited disorder with multi-systemic effects that impact the skeletal muscles, eyes, heart, skin and gastrointestinal, endocrine, respiratory, and central nervous systems. DM is divided into two subtypes: DM1 can present from early childhood through adulthood and also has a congenital form (cDM) while DM2 typically manifests during mid-adulthood. Both forms are progressive with no approved treatments, and unmet need for disease-modifying therapies remains high. This study interrogated health insurance claims data to explore the clinical experience, healthcare resource utilization (HCRU), and all-cause costs for DM.A total of 8541 patients with DM and 242 patients with cDM and their matched controls were selected from a database of over 200 million claimants. HCRU and all-cause costs, including pharmacy, outpatient, and inpatient services, were analyzed across four years in 12-month follow-up periods. Mean all-cause costs per DM patient were high in each of the four periods  and showed a steady increase from 13 to 23 months on, while the control group mean costs declined from $9671 in the first 12 months after the index event, to approach the US population average ($5193) over time. For cDM, the highest mean costs were in the first 12-months , and remained high  across all subsequent periods, while control mean costs approached $0. For DM and cDM, HCRU was higher compared to controls across all study periods and all-cause healthcare costs were mostly driven by inpatient and outpatient encounters. Analysis of all diagnosis codes over the study period (comorbidities) demonstrated an elevated comorbidity profile consistent with the clinical profile of DM.This study is among the first to utilize claims data to increase understanding of the clinical experience and health economic outcomes associated with DM. The markedly elevated HCRU patterns and comorbidity profile presented here add to the broad body of scientific and clinical knowledge on DM. These insights can inform clinical care and support the development of disease modifying and/or symptom-targeting therapies that address the multi-systemic, progressive nature of DM.The online version contains supplementary material available at 10.1186/s13023-022-02241-9. Myotonic dystrophy (DM) is a complex, multi-systemic neuromuscular disease that is characterized by delayed relaxation of skeletal muscles (myotonia) and progressive muscle weakness and degeneration (muscular dystrophy) –4. It isWhile past research has demonstrated a high disease burden for patients with DM, the healthcare resource utilization (HCRU) and healthcare costs for this understudied population are relatively unexplored , 18, 19.–70% of US-based physician activity, and approximately 85% of dispensed prescriptions for all pharmacies in the US. Similar methods have been used to characterize the HCRU and costs associated with medical management of spinal muscular atrophy (SMA), ankylosing spondylitis, and Duchenne muscular dystrophy (DMD) , physician office visits , ER visits , and a remarkably increased number of outpatient visits . A greater proportion also had ≥ 1 hospitalization .Similarly, patients with cDM compared with matched control patients had significantly increased mean prescription fills [15.45 (17.03) vs 7.30 (7.20); The pattern of significant difference in HCRU between DM and non-DM and cDM and non-cDM cohorts is consistent across all four follow-up periods. Additional HCRU in each of the above-listed categories is shown for DM and cDM across all study time periods in Additional file Using claims data derived from the IQVIA New Data Warehouse, this study found that the total all-cause costs and HCRU per patient per year were considerably higher for patients with DM and cDM compared with matched controls. This longitudinal, retrospective, real-world study generated a wealth of data, both in size and number of variables. It is among the first to specifically examine HCRU and total all-cause costs in DM in detail and make efforts to elucidate the experience of those congenitally affected (cDM) , 24. By The mean all-cause costs per patient per year averaged $15,852 for the DM cohort compared to $6688 for the non-DM cohort. This elevated cost is consistent with the findings of Larkindale and colleagues, who found that direct medical costs per patient with DM per year was $17,451 . For comOverall costs for the DM cohort are markedly elevated compared to controls across all four study time periods. The relatively high costs for both the DM and non-DM cohorts in the first follow-up period (< 12 months) can be attributed, at least in part, to the increased likelihood that the incident case is associated with an acute, cost-incurring medical event. In subsequent periods however, a trajectory of increasing costs for the DM cohort that reflects the chronic and progressive nature of DM is apparent, while costs for the control group steadily approach the US national average over time.Total all-cause costs for the cDM cohort compared to their control group follow a similar pattern, in that there is an increase in costs in both the cDM and control group in the < 12 months follow up period; however, for cDM initial costs are extraordinarily high, reflecting the severe nature of cDM and the intensive clinical care interventions required to treat these neonates during this early phase of their lives. In subsequent time periods, the all-cause costs for the cDM cohort decrease, but remain elevated relative to the non-cDM cohort (which decline and approach zero), reflecting the considerable ongoing needs associated with this most severe form of myotonic dystrophy.Results from this study revealed the following drivers of HRCU among patients with DM or cDM: outpatient, physician office, and ER visits; inpatient hospitalizations; and increased use of prescription medications. For example, the mean number of outpatient visits was 26.78 for the DM cohort vs. 12.23 for controls and the percentage of patients with at least one hospitalization was 2.9% vs. 1.9% in the first 12 months Table  and for or ICD-10 code) represent a conservative estimate of true incidence/prevalence. For the purposes of analysis, ICD-9 codes were not combined with corresponding/equivalent ICD-10 codes to avoid double counting codes for cases that crossed the code transition date, which would result in an overestimation. While the relative frequency (compared to controls) reported for any given ICD-9 or ICD-10 code is reliable, the absolute frequency is likely an underestimate.In this study, numerous comorbidities were identified at varying frequencies and levels of significant difference compared to controls that illustrate the disease burden in DM. The detailed comorbidity findings are not presented here but the data tables are available for further analysis upon request. The most impactful comorbidities (defined above) varied with DM type and also highlighted underappreciated symptoms, which not only can dramatically affect quality of life, but can interfere with functions important in work and/or academic performance . It should also be noted that the comorbidities identified in this study negatively affected the patient to a degree that prompted the patient to present to a medical facility for care or came to the attention of the treating clinician. During the first follow-up year, GI system symptoms 14.4%), cardiac dysrhythmias 12.6%), and organic sleep disorders (11.7%) were the most prevalent comorbidities among patients in the DM cohort . Table S1. Baseline Demographic Characteristics (post-match). Table S2. DM Cohort Most Impactful Comorbidities. Table S3. cDM Cohort Most Impactful Comorbidities. Table S4. Total HCRU & All-cause Costs for DM Cohort over 12-, 13-23, 24-35, 36-47 & 48+ Month Follow-Up Periods. Table S5. Total HCRU & All-cause Costs for cDM Cohort over 12-, 13-23, 24-35, 36-47 & 48+ Month Follow-Up Periods.


Biomphalaria pfeifferi (BP) and Bulinus globosus (BuG), two of the world’s most important schistosomiasis vectors in Africa. Sequences from 26 snails generated using Illumina Hi-Seq or 454 sequencing were assembled using Trinity and CAP3 and putative virus sequences were identified using a bioinformatics pipeline. Phylogenetic analyses were performed using viral RNA-dependent RNA polymerase and coat protein sequences to establish relatedness between virus sequences identified and those of known viruses. Viral sequences were identified from the entire snail holobiont, including symbionts, ingested material and organisms passively associated with the snails. Sequences derived from more than 17 different viruses were found including five near full-length genomes, most of which were small RNA viruses with positive sense RNA genomes  and some of which are likely derived from adherent or ingested diatoms. Based on phylogenetic analysis, five of these viruses (including BPV2 and BuGV2) along with four Biomphalaria glabrata viruses reported previously, cluster with known invertebrate viruses and are putative viruses of snails. The presence of RNA sequences derived from four of these novel viruses in samples was confirmed. Identification of the genome sequences of candidate snail viruses provides a first step toward characterization of additional gastropod viruses, including from species of biomedical significance.Schistosomiasis, which infects more than 230 million people, is vectored by freshwater snails. We identified viral sequences in the transcriptomes of  Biomphalaria and Bulinus transmit human intestinal and urinary schistosomiasis, respectively. Snails serve as the obligatory hosts of the asexually-reproducing larval stages of schistosomes that can produce hundreds to thousands of human-infective cercariae per day. Some infected snails can continue to release cercariae into the water for several months  (Paenibacillus)  , and snaacillus)  have potacillus) , we sougBiomphalaria glabrata were isolated by analyzing available RNA sequence data for this Neotropical snail  and examined the RNA sequences for evidence of the presence of viruses. We identified sequences derived from multiple viruses that based on phylogenetic analysis, are predicted to infect and replicate in the snail. We also identified sequences from numerous other viruses including four near full length genomes, that are likely infectious to diatoms and other aquatic microorganisms associated with the snail.Here we report use of our bioinformatics pipeline , to idenBiomphalaria pfeifferi (BP) and Bulinus globosus (BuG), were collected from Kenya. BP snails . Complementary DNA libraries were paired-end sequenced (2 × 50 base reads) on a HiSeq2000 instrument . A total of 20 to 30 million reads of 50 nucleotides were generated from each individual snail with three sequencing replicates per sample .Methods for RNA extraction from snail samples  and highr sample . Sequenc5 and 26  on the 4http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). When necessary, bases with low quality scores  were trimmed using Fastx-toolkit v0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/). Viral genome-derived sequences were either assembled from sequence data from individual snails, or from the combined sequence data from three replicate snails  was used for mapping of reads to target assembled sequences.The quality of sequencing reads was examined using FastQC v0.11.3  according to the manufacturer’s directions. Primer design was based on the assembled viral sequences . PCR washttp://iresite.org/). Sequences of conserved domains were extracted and used for phylogenetic analysis. The domain sequence was aligned using MAFFT (with MAFFT-auto and default parameters) (http://www.mbio.ncsu.edu/BioEdit/page2.html).Conserved domains of the viral sequences identified were used for phylogenetic analysis. The conserved domains of RNA-dependent RNA polymerase (RdRP) and capsid proteins or putative capsid sequences were identified by BLAST domain search against the NCBI nr database . Internaameters) . Phylogeameters)  with 10,ameters) . The besameters) . The phyameters) . GeneralThe number of contigs derived from putative viral sequences from the snail samples is shown in Biomphalaria pfeifferi-associated virus 1, BPV1: GenBank accession number MT108488; Marine RNA virus JP-A (MRNAVJPA) . The second closest hit was Rhizosolenia setigera RNA virus 01 (RhsRNAV01)  of dicistroviruses were identified in ORF1  assembled by Trinity. One near complete small RNA viral genome (named RNAVJPA) , a MarnasRNAV01) , with 27 in ORF1 . ORF2 en in ORF1 .Biomphalaria pfeifferi-associated virus 2 (BPV2: GenBank Accession No. MT108489), Biomphalaria pfeifferi-associated virus 3 (BPV3: GenBank Accession No. MT108490) and Biomphalaria pfeifferi-associated virus 4 (BPV4: GenBank Accession No. MT108491), with genomes of at least 8,491, 8,255 and 8,253 nt, respectively , which was discovered from Antarctic lakes, but for which the host is unknown . ProteinMarnavirus. The sequence identities were ~35% between the corresponding ORF polyproteins of BPV3 and MRNAVJPB. ORF2 of BPV3 encodes 942 aa, which is the longest ORF2 of the dicistrovirus-like sequences discovered from snails. Three CP domains were identified in the ORF2 polyprotein of BPV3, Rhv_like CP domain, VP4, and the CrPV_capsid domain, which also contained conserved sequences of an Rhv_like domain. For all three dicistrovirus-like viruses, translation of BPV2 ORF2 is expected to be mediated by an internal ribosomal entry site (IRES) (The top hits for the ORF1 and ORF2 polyproteins of BPV3 were both from Marine RNA virus-JP B (MRNAVJPB) , which ie (IRES) .Marnavirus, Heterosigma akashiwo RNA virus (HaRNAV)    . The N-tke virus  with 27%ke virus .In addition to the four near complete full-length SRV+ genome sequences described above from BP, 44 putative SRV+ contigs were identified, ranging from 203–3,624 nt . Among tChaetoceros salsugineum, relatively short sequences with homology to the SRV+ Picalivirus D  and a plant virus, Currant latent virus (BPVS54). In addition, there were three contigs  that hit hypoviruses. While the top hit of BPVS45 is Rosellinia necatrix fusarivirus 1, this contig also hit hypovirus sequences. Hypoviruses are double-stranded RNA (dsRNA) viruses, which are only known to infect Fungi (http://ictvonline.org/virusTaxonomy.asp). Protein sequence identities of the four putative hypoviral sequences to known hypoviruses were 26–50%.Nine contigs of 206–2,969 nt had homology to several viruses , includiivirus D  (BPVS51)Bulinus globosus-associated virus 1 (BuGV1: GenBank Accession No. MT108492) and a Nora virus-like virus  were identified. The other putative viral contigs are listed in Fewer viral sequences were assembled from the three sets of BuG sequence data. Only nine contigs derived from viral sequences were identified by BLASTx. From the nine virus-related contigs, one near complete SRV+ genome sequence, named Similar to BPV1, BPV2 and BPV3, BuGV1 is dicistrovirus-like with 8,528 nt of assembled sequence . The nonDrosophila that encodes four potential ORFs , suggesting these two contigs may be derived from the same virus (BuGV2). Nora virus is an SRV+ of ial ORFs  Fig. 4)Drosophilial ORFs . BuGV2 cAs BPV2-derived sequence was present in all but one of the sequencing datasets used for virus sequence identification , we firsTo determine the distribution of virus-derived sequences among the snail samples, we examined the viral sequences by mapping the Illumina sequencing reads (50–100 nt) to the five near complete  and to sBiomphalaria glabrata virus 1, BGV1; Biomphalaria glabrata virus 2, BGV2; Biomphalaria glabrata virus 3, BGV3; and Biomphalaria virus 4, BiV4) identified from RNA sequence data for B. glabrata  were extracted, aligned and used for construction of a phylogenetic tree. This RdRP-based phylogenetic analysis clustered the snail viruses into three groups , Bacillarnaviruses (with marine diatom hosts) and insect small RNA viruses . There was no similarity to SRV+ infecting marine mammals,  virus 1 . The RdR virus 1 . To anale groups : First, In addition to eight complete small RNA viral CP, five sequence fragments  encode near full-length putative CP >90%;  and wereTaken together, both RdRP and CP sequences suggest that BPV1, BPV3, BPV4 and BuGV1 are aquatic viruses, while BPV2 is dicistro-virus like. While the CP of BGV1 and BGV3 place these viruses as iflavirus-like, this association is unclear from analysis of RdRP sequences.B. pfeifferi and B. globosus. In so doing, we have characterized one aspect of the complex microbiome encountered by schistosome parasites in their snail vectors , or encoded a single polypeptide , in which the nonstructural proteins are located at the N-terminus, and the structural proteins at the C-terminus  and other insect or marine RNA viruses.The majority of viruses identified from the two species of snails reported here, along with viruses identified previously from glabrata  are simiglabrata . Howeverglabrata , Fig. 2)glabrata . The genhttp://www.iresite.org/). Similar to insect SRV+, SRV+ from aquatic environments usually have four capsid subunits (VP1–VP4) that are generated on proteolytic cleavage of the structural polyprotein. The subunit arrangement of the viral structural proteins of snail and diatom infecting SRV+ is similar to that observed in insect infecting dicistroviruses, in that the smallest VP4 is not the first subunit in the structural polyprotein  of BPV1, BPV2, BPV3 and BuGV1 by IRESite , insect SRV+  and SRV+ isolated from aquatic arthropods (shrimp and crab). Few BLAST hits were from vertebrate SRV+, e.g., Picalivirus and Calicivirus, commonly found in sewage-derived RNA . BuGV1 amplification product was amplified only from BuG samples. Lane numbers correspond to sample numbers in Click here for additional data file.10.7717/peerj.12290/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12290/supp-3Supplemental Information 3Fifty-four partial viral sequences were identified from BP, and three from BuG. Sequence lengths and details of virus proteins with highest similarity based on BLAST analysis are shown. Accession numbers for all sequences are provided.Click here for additional data file.10.7717/peerj.12290/supp-4Supplemental Information 4Click here for additional data file.


Merkel cell carcinomas (MCCs) of unknown primary are defined as deep-seated tumors without an associated cutaneous tumor. Although the distinction has important clinical implications, it remains unclear whether these tumors represent primary tumors of lymph nodes or metastatic cutaneous primaries. We compared the immunohistochemical profiles of four groups of Merkel cell carcinomas (virus-positive and virus-negative unknown primary tumors and virus-positive and virus-negative cutaneous tumors) and performed molecular studies on the unknown primary tumors. Virus-positive and virus-negative Merkel cell carcinomas of unknown primary (MCC-UPs) exhibited an immunoprofile similar to virus-positive and virus-negative primary cutaneous MCCs, respectively. Similar to primary cutaneous Merkel cell carcinomas, virus-negative unknown primary tumors exhibited UV signatures and frequent high tumor mutational burdens, whereas few molecular alterations were noted in virus-positive tumors. Although additional studies are warranted for the virus-positive cases, our findings are supportive of a cutaneous metastatic origin for virus-negative Merkel cell carcinomas of unknown primary.Background: Merkel cell carcinomas of unknown primary (MCC-UPs) are defined as deep-seated tumors without an associated cutaneous tumor. Although the distinction has important clinical implications, it remains unclear whether these tumors represent primary tumors of lymph nodes or metastatic cutaneous primaries. Methods: We compared the immunohistochemical profiles of four groups of MCCs (Merkel cell polyomavirus (MCPyV)-positive UP, MCPyV-negative UP, MCPyV-positive known primary (KP), and MCPyV-negative KP) using B-cell and pre-B-cell markers, cell cycle regulating proteins, follicular stem cell markers, and immune markers, and performed next generation and Sanger sequencing. Results: Virus-positive and virus-negative MCC-UPs exhibited an immunoprofile similar to virus-positive and virus-negative primary cutaneous MCCs, respectively. MCC-UP tumors (both virus-positive and -negative) were immunogenic with similar or even higher tumoral PD-L1 expression and intratumoral CD8 and FoxP3 infiltrates in comparison to MCPyV-positive cutaneous tumors. In addition, similar to primary cutaneous MCCs, MCPyV-negative MCC-UPs exhibited UV signatures and frequent high tumor mutational burdens, whereas few molecular alterations were noted in MCPyV-positive MCC-UPs. Conclusions: Our results showed distinct UV-signatures in MCPyV-negative tumors and high immunogenicity in MCPyV-positive tumors. Although additional studies are warranted for the MCPyV-positive cases, our findings are supportive of a cutaneous metastatic origin for MCPyV-negative MCC-UP tumors. TP53) and RB transcriptional corepressor 1 (RB1) mutations [Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine tumor with high mortality 33–46%) ,2,3,4,5.3–46% ,2,utations ,10. Approximately 4% of all MCCs present as deep-seated tumors in the parotid, axillary, or inguinal region with no overlying skin involvement or a history of primary tumor . MCC of Despite a clear molecular delineation between the MCPyV-positive and MCPyV-negative cutaneous MCCs, little is known about the biology of MCC-UP. The distinction between whether these tumors represent primary tumors or metastases is important for the development of relevant preclinical models and would affect treatment strategy. In order to investigate the potential cell-of-origin and possible molecular therapeutic targets, we compared the immunohistochemical profiles of the four groups of MCCs , and MCPyV-negative KP) using B-cell and pre-B-cell markers , cell cycle regulating proteins , follicular stem cell markers , and immune markers . We also analyzed the molecular profiles of MCPyV-positive UPs versus MCPyV-negative UPs by next generation sequencing (NGS) and targeted Sanger sequencing.p = 0.037) and CK15 (p = 0.014) expression than MCPyV-positive UP cases.The ages of the 27 patients  with MCC-UPs ranged from 49 to 95 years . Although a significant history of immunosuppression was not noted in these patients, comorbidities of prostatic carcinoma and melanoma were documented in three and four patients, respectively. The tumor was within lymph nodes of the parotid gland (15 cases), the pelvic/groin region (7 cases), the submandibular gland (4 cases), and the axilla (1 case). Their sizes ranged from 1.0 to 9 cm . Overall, 6 patients received surgery, 6 received surgery and radiation, and 15 received surgery, radiation, and chemotherapy (12 with carboplatin/etoposide and 3 with pembrolizumab). Follow-ups ranged from 0 to 201 months . Death was documented in 13/27 (48%) of patients. The immunohistochemical results of MCC-UP cases versus MCPyV status are summarized in The clinicopathologic variables of the primary cutaneous MCCs are summarized in p = 0.0002) and associated keratinocytic neoplasms (p = 0.0001) was significantly correlated with MCPyV-negative status.In total, 64 tumors (48%) were from the head and neck region and 70 (52%) were from other sites. The median tumor size and tumor thickness were 20 mm (range: 2 to 125 mm) and 10 mm (range: 1 to 55 mm), respectively. Mitoses per squared millimeter ranged from 1 to over 100 . Ulceration, necrosis, perineural invasion, and lymphovascular invasion were present in 45 (34%), 44 (33%), 13 (10%), and 64 (48%) cases, respectively. The presence of epidermotropism (p = 0.012) with the worst survival noted in the virus-negative KP group (p = 0.44). MCC-UP tumors with high intratumoral FoxP3+ and CD8+ infiltrates exhibited better OS   . There was no survival difference observed in the MCC-UP group with respect to MCPyV status, PD-L1, IDO1, and TdT . No significant associations between intratumoral FoxP3+ infiltrate (p = 0.42 and 0.19) and CD8+ infiltrate (p = 0.94 and 0.23) versus OS were observed in virus-positive and virus-negative KP groups, respectively.Kaplan–Meier curves demonstrated significant differences in the overall survival (OS) among the three groups  (KP group A. Kaplanctively) B. When octively) , whereasp-values with false discovery rate corrections are summarized in p = 0.026 and 0.088, respectively) (p = 0.0096) , and in virus-positive KPs versus virus-negative UPs and virus-negative KPs   A,B. Nucl 0.0096) C. Rb expctively) D. There p = 0.017, 0.009, and 0.017, respectively) . A sequential decrease in tumoral PD-L1 expression, combined tumoral PD-L1 and high intratumoral FoxP3+ cell count, combined tumoral PD-L1 and high intratumoral CD8+ cell count, and combined high intratumoral CD8+ and FoxP3+ cell counts was observed in the virus-positive UP, virus-negative UP, virus-positive KP, and virus-negative KP groups   E. Althouctively) F. We obsctively) .TP53 and RB1 mutations and/or insertions/deletions with C > T/G > A and CC > TT/GG > AA transitions (CC > TT highly suggestive of UV-induced mutation) were detected in all six virus-negative MCC-UPs  in cutaneous MCC, were significantly correlated with an MCPyV-negative status ,27. ThesIn our series, virus-positive and virus-negative MCC-UPs exhibited an immunoprofile similar to virus-positive and virus-negative cutaneous MCCs, respectively, which is highly suggestive that UP tumors represent metastases of cutaneous primary. The overexpression of TdT and Pax5 as well as the up-regulation of Rb protein has been shown to significantly correlate with MCPyV positivity in cutaneous MCCs . On the p = 0.036) when the entire primary cutaneous MCC group (virus-positive and virus-negative KP) was analyzed (unpublished data). While the CD8+ infiltrate was examined for whole tumor sections in prior studies [A strong immune response has been shown to correlate with a better outcome for MCC patients . Several studies ,31,34, w studies ,33,34. I studies  reportedIn our series, MCC-UP tumors (both virus-positive and -negative) were more immunogenic than their cutaneous counterparts. Of interest, the virus-positive UP group in our study appears to be the most immunogenic, with the highest frequency observed in the virus-positive UP group with respect to tumoral PD-L1 expression and in combination with high intratumoral CD8+ and FoxP3+ infiltrates. This immunoprofile observed in MCC-UPs is more in line with virus-positive MCC-KPs than virus-negative KPs. As noted in our and other studies, MCC-UP has a lower association with MCPyV than MCC-KP cases (25% versus 63%) . Our finTP53, RB1, and PIK3CA [NF1, PIK3CA, PTEN), ARID1A, and DNA repair genes . Our and prior studies suggested that it is likely that a combination of either high TMB or viral antigens and increased immunogenicity might account for the regression of primary tumors and an improved prognosis in MCC-UP cases [Sequencing analyses by NGS and Sanger performed in our study showed that MCC-UP is comprised of two tumor groups with mutually exclusive segregation of UV or MCPyV pathways, as is described in recent studies of MCC-UP ,16 and md PIK3CA ,20,21,39UP cases ,16.Our study has several limitations, including its retrospective and multicenter nature, the small MCC-UP sample size, because of the rarity of MCC-UP, and only having 12 patients with stage III in the MCC-KP group. Due to the poor DNA quality of formalin-fixed paraffin-embedded tissues in older cases, we could perform NGS assays only on recent cases. Nevertheless, our study represents the first immunohistochemical comparison of the subgroups of MCC: virus-positive and virus-negative UP, and virus-positive and virus-negative KP groups.The study was approved by Institutional Review Boards review of various institutions. A retrospective review of the pathology archives at six clinical institutions in Poland, Taiwan, and the United States from 1990 to 2020 yielded 134 primary cutaneous MCCs and 27 MCCs in lymph nodes of the parotid, axilla, and inguinal regions with no known primary. The diagnoses were histopathologically confirmed by the contributing pathologists and the corresponding author (MPH). All the tumors had diagnostic features of MCC, including characteristic histologic appearances and expression of CK20 and neuroendocrine markers . Neurofilament, CK7, or other CKs , and EMA were assessed in a subset of cases. Thyroid transcription factor 1 (TTF1) was assessed in the majority of cases. Medical records, including clinical notes, pathology, and radiology reports, were reviewed to exclude the possibility of metastatic neuroendocrine carcinomas and to document clinical parameters, including age, lesion site, history of immunosuppression, stage, and disease status over time and at last follow-up  .Tissue microarrays composed of two 2 mm tissue cores from each tumor were constructed. Immunohistochemical studies were performed on 5-micrometer-thick tissue sections using a Bond 3 automated immunostainer , with primary antibodies against MCPyV large T-antigen , TdT , Pax5 , CK15 , CK19 , p53 , RB , PD-L1 , IDO1 , CD8 , and FoxP3 .H-score ((percentage at 1+) × 1 + (percentage at 2+) × 2 + (percentage at 3+) × 3), which combines the intensity and the percentage of positive cells into a combined score. The median H-scores of 20, 70, 190, 155, and 80 were used as cutoff values for TdT, Pax5, p53, Rb, and CK19, respectively. Greater than 1% of tumor cells with membranous PD-L1 staining was considered positive. Any staining with CM2B4 and CK15 was considered positive. The absolute cell counts of CD8+ and FoxP3+ lymphocytes within the tumor were assessed in three consecutive images captured at 40× magnification of the hotspot, the area with the densest labeling index. The scores were dichotomized into high and low using the median as the cutoff value.Scoring was done by two independent authors blinded to clinical outcomes. Tumor cells with positive CM2B4 nuclear staining of any intensity were considered positive. Scoring of CM2B4, TdT, Pax5, CK15, CK19, p63, p53, RB, and IDO1 immunostains was done using the TP53), RB transcriptional corepressor 1 (RB1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and the presence of MCPyV were performed on DNA extracted from 27 formalin-fixed paraffin-embedded MCC-UPs by polymerase chain reaction (PCR) and sequencing with specific primers using previously reported methods NGS-based molecular analyses were successfully performed in 11 cases. The assay utilizes anchored multiplex polymerase chain reaction (PCR) to detect single nucleotide variants (SNV) and small insertion/deletions (indel) in genomic DNA using NGS . A sequep-values were adjusted with the false discovery rate correction. All analyses and plots were performed using the R statistical package [p-value of less than 0.05 was considered statistically significant.The statistical association between MCPyV T antigen expression (detected by the CM2B4 antibody) and other proteins and clinicopathologic features  were evaluated by Fisher’s exact tests. American Joint Committee on Cancer stage versus MCPyV status was calculated using the Cochran–Armitage test. Overall survival (OS) was defined as the number of months from the initial diagnosis to the patient’s death by any cause. Kaplan–Meier plots and log-rank tests were created to visually assess the differences in the OS between subgroups. Box plots were used for the comparison of the expression of various immunostains. An assessment of the statistical significance of the differences between the groups was performed using Anova (for continuous expression) or the chi-squared test (for positive/negative markers). In a post-hoc analysis, Tukey’s test was applied to identify pairwise differences. Because of multiple testing,  package . A two-tIn conclusion, we have comprehensively compared subtypes of MCC (UP and KP with respect to MCPyV status) and demonstrated significant immunophenotypic, immunologic, and molecular similarities between virus-negative MCC-UP and MCC-KP as well as virus-positive MCC-UP and MCC-KP. Our results showed distinct UV-signatures in MCPyV-negative tumors and high immunogenicity in MCPyV-positive tumors. Although additional studies are warranted for the MCPyV-positive cases, our findings are supportive of a cutaneous metastatic origin for MCPyV-negative MCC-UP tumors.


The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation. Hematopoiesis occurs in the bone marrow (BM) as a result of the crosstalk between, among others, mesenchymal and hematopoietic stem cells. Their interaction occurs in niches in which complex networks are built upon concerted cell-to-cell architectures. These compartments have been delineated in animal models at single-cell resolution , revealiAlterations in these microenvironments, e.g. by viral infections, may result in multiple pathological conditions. Indeed, persistent viruses can perturb the homeostasis, either by cytotoxic damage of the niche´s structural integrity, transformation of the signaling repertoire, or interference with the cellular trafficking . HoweverMoreover, while most studies have focused on single viruses , a comprParvoviridae, Herpesviridae, Polyomaviridae, Hepadnaviridae, Anelloviridae, and Papillomaviridae families. To the best of our knowledge, this is the most comprehensive assessment of viral DNAs present in the BM to date.In the present study, we systematically investigated the genomic prevalence and copy numbers of altogether 38 clinically significant DNA viruses in the BM of 27 recently deceased individuals. Included were members of the The study cohort consisted of 27 deceased individuals of Finnish origin. The age range of the study subjects was 36 to 85 years (mean 68) and the postmortem interval on average 8 days Figure 1The BM was collected from the diaphysis of femoral bone and the DNA extracted with QIAamp DNA Mini Kit (Qiagen) as per manufacturer protocol. The quality and integrity of total DNA in each sample was confirmed with a Genomic DNA Reagent Kit in a LabChip GX Instrument.To avoid contamination, strict precautions were taken throughout the experiments including the use of single-use disposable plastics and filter tips, the handling in hoods dedicated exclusively to nucleic acid work, and the use of negative controls through all steps. The reaction mixes, DNA extracts, plasmid controls, and amplification reactions, were each handled in separate rooms.The viral DNAs were analyzed by targeted enrichment followed by sequencing in NovaSeq 6000 (Illumina), as well as by quantitative PCRs.For the enrichment, a customized panel of biotinylated RNA oligonucleotides (Arbor Biosciences) was used. The viruses targeted were parvovirus B19 (B19V), human bocaviruses 1-4 (HBoV1-4), cutavirus, human papillomavirus (HPV) types 2, 6, 11, 16, 18, 21, and 45, human polyomaviruses (HPyV) 1-13, simian virus 40, human herpesviruses (HHV) 1-8, hepatitis B virus (HBV), torque teno virus (TTV) and variola virus (minor and major). A complete list of the reference strains used for the design of the RNA oligonucleotides, as well as the details of the NGS methods, are presented in . In brieThe consensus, as well as single sequences (when in low coverage), were confirmed by BLAST (NCBI).To confirm the NGS findings, validated in-house quantitative PCRs were used for the detection of B19V , Merkel We conducted a systematic analysis of viral DNAs present in the bone marrow by two complementary approaches, NGS and qPCR.We first analyzed the integrity of the total genomic DNA of each sample to control for bias in the prevalences and loads detected by qPCR. Based on the size distribution, the total DNA was of high quality and non-fragmented, except for one sample, taken 30 days post-mortem, exhibiting a wider range of fragment sizes Figure 2We detected viral DNA in 88.9% of the samples, comprising altogether 11 distinct virus types Table 1.On average, we identified two virus types per individual, the most common co-detections being of B19V and TTV, in 44.4% of the BMs Figure 1When normalized to copies per million cells, the highest quantities were of B19V and TTV with a median of 7.9E3 and 2.8E5, respectively, to be followed by EBV with 9.1E1 copies. VZV, HCMV, HHV-6B, HHV-7 and HBV had respectively 2.5E1, 1.0E2, 3.8E1, 3.4E2 and 1.2E3 copies. In general, the quantities of most viral DNAs were very low, speaking against active replication.Via in-solution hybridization, we captured and reconstructed 11 viral genomic sequences, with a minimum breadth coverage of 15% Figure 3Moreover, albeit in low coverage, we manually and visually confirmed by BLAST each of the unique sequences (mean length 100 nucleotides) specific for the following viruses: VZV , EBV , HHV-7 , MCPyV , TTV , B19V , and HBoV-1 .Because of our cohort’s advanced age and the sampling location, a higher proportion of mesenchymal cells are to be expected. We compared the sequences of the viruses found in the BM with those detected in the same individuals´ external surface of the femoral bone . We founThe reconstructed consensus sequences of B19V have been deposited in GenBank with accession numbers: MT988397-MT988403.A comprehensive analysis of the virus types residing in the human bone marrow is lacking. Yet, this knowledge is essential for assessment of the impact of viral DNA persistence on the marrow’s integrity and on the emergence of many pathological conditions. Since the BM homeostasis is founded on many precise regulatory networks, any alterations resulting from single or cumulative effects of multiple viruses can foreseeably have profound effects on its output and function.We evaluated the prevalences of viral DNAs residing in the bone marrow and found 11 different virus types, in copy numbers compatible with latency.Remarkably, among the viral genomes identified, we found HPV-31 in a male individual aged 68, who died of atherosclerotic heart disease. Since infections by this oncovirus are normally self-limited and restricted to epithelial surfaces, the detection of its DNA in BM is perplexing. Other HPV types have been reported in BM by Payne et al. , albeit Another interesting finding was that of the genome of MCPyV, likewise of mucocutaneous tropism and oncogenic potential. We detected it in three individuals, two of whom were also positive in the bone. MCPyV DNA in BM was reported by Matsushita et al. , who fouImportantly, advanced stages of malignant transformation by both papillomaviruses and Merkel cell polyomavirus can present with manifestations in bone . While nin vitro a wide range of constituent and memory cells have been shown permissive for one or more  can be found in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Helsinki and Uusimaa Hospital District . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MT, AS, KH, and MP designed the study. MT performed the experiments. MT, DP, and MP analyzed the data. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Finnish Medical Society (MP), the Jane and Aatos Erkko Foundation (KH), the Finnish Cultural Foundation , the Sigrid Jusélius Foundation (KH), the Life and Health Medical Foundation , the Magnus Ehrnrooth Foundation , Finnish Society of Sciences and Letters , the Kone Foundation (AS), the Research Funds of University of Helsinki and Helsinki University Hospital (KH), Finska Läkaresällskapet , Fundação para a Ciência e a Tecnologia, I.P., under the Scientific Employment Stimulus - Institutional Call - CI-CTTI-94-ARH/2019 (DP).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The structural study of icosahedral viruses has a long and impactful history in both crystallographic methodology and molecular biology. The evolution of the Protein Data Bank has paralleled and supported these studies providing readily accessible formats dealing with novel features associated with viral particle symmetries and subunit interactions. This overview describes the growth in size and complexity of icosahedral viruses from the first early studies of small RNA plant viruses and human picornaviruses up to the larger and more complex bacterial phage, insect, and human disease viruses such as Zika, hepatitis B, Adeno and Polyoma virus. The analysis of icosahedral viral capsid protein domain folds has shown striking similarities, with the beta jelly roll motif observed across multiple evolutionarily divergent species. The icosahedral symmetry of viruses drove the development of noncrystallographic symmetry averaging as a powerful phasing method, and the constraints of maintaining this symmetry resulted in the concept of quasi-equivalence in viral structures. Symmetry also played an important early role in demonstrating the power of cryo-electron microscopy as an alternative to crystallography in generating atomic resolution structures of these viruses. The Protein Data Bank has been a critical resource for assembling and disseminating these structures to a wide community, and the virus particle explorer (VIPER) was developed to enable users to easily generate and view complete viral capsid structures from their asymmetric building blocks. Finally, we share a personal perspective on the early use of computer graphics to communicate the intricacies, interactions, and beauty of these virus structures. The Protein Data Bank (PDB) has played a pivotal role for disseminating virus structure atomic coordinates in a manner that has been useful for virologists and other noncrystallographers in recent years. Achieving this utility has had a number of iterations with a variety of contributions from structural virologists and the developers of graphics programs. Indeed, there has been a refreshing level of cooperation among a number of participants that has brought the enterprise to its current state of effectiveness. In this contribution the early developments of virus crystallography are briefly presented, the progress in the ease of use of the PDB data resource for virologists is described, then the history of some features of structural virology as documented in icosahedral virus PDB submissions since 1984.The structural study of icosahedral viruses has a long and impactful history in propelling forward both crystallographic methodology and the understanding of molecular biology. In the 1930s, Bernal and Fankuchen made the first crystallographic studies of a spherical plant virus, Tomato Bushy Stunt Virus (TBSV) . That saIt wasn’t until the late 1970s that computers and crystallographic computing methods enabled atomic-level resolution of structures as large as intact virus particles. The 5.5 Å resolution crystal structure of TBSV, determined in 1977 in the Harrison Lab . The sub32 symmetry site, so all but the 5fold icosahedral axes were part of the crystal lattice. A pentamer of trimers  were deposited. SBMV crystallized in a rhombohedral space group with the particle center at a 23 symmetry site implying 2 pentamers of trimers in the crystallographic asymmetric unit and 30 subunit coordinate sets deposited. Since NCS was critical in phase determination and improvement, it was noteworthy that TBSV had fivefold NCS and SBMV tenfold NCS. This specified that the SBMV crystal form had 1.4 times the phase improvement power of the TBSV crystal form since phase enhancement goes as the square root of the NCS.TBSV  from theBoth structure determinations required numerous person-years of effort  and in the end there were two Nature papers ; second,Box 1Translating virus coordinates into useful 3d graphics was first accomplished by one of us (AJO) with TBSV along with other efforts in the use computer graphics to generate 3d models of viruses. A narrative history of the development of the first 3d tour of a virus particle as well as other virus animations is provided in The ssRNA picornaviruses, Poliovirus (PV)  and Humai.e., ionic, hydrophobic, etc.)  . These wc, etc.) , 26, 27.c, etc.) .Figure 3Following the first four virus structures deposited to the PDB, the pace picked up . By 1990A variety of technology advances resulted in 59 virus entries by 1995. The first ssDNA virus, the bacteriophage phi X 174, was submitted in 1991 and displayed the jellyroll fold in the two different major gene products that formed the icosahedral asymmetric unit of the T = 1 particle . The mamThe count of icosahedral virus particle deposits was at 134 by 2000 and some viruses of exceptional importance were deposited between 1995 and 2000. The transcriptionally active Blue Tongue Virus core was among those with the greatest impact. This dsRNA virus in the family reovirus has a T = 13 l outer shell formed by a two-domain protein with a helical interior and a protruding viral jellyroll on the outside. Interior and adjacent to the RNA was a T = 1 core formed by 120 copies of a predominantly helical protein with a totally different fold from other viral capsid proteins. The deposition in 1998 set a number of records for a virus. At 700 Å in diameter it was the largest virus in the PDB; it had the largest T number at 13; and it was the first to have shells with two different symmetries . Two yeal Hong Kong 97 VLP  that had a subunit fold similar to the mammalian adenoviruses. The PDB had become an exceptionally valuable resource for understanding virus particle evolution.Approximately 100 icosahedral virus structures were submitted to the PDB between 2001 and 2005. A quarter of these were structures based in part on cryoEM density. The “gold standard” in this time frame for cryoEM was sub-nanometer resolution structures, although many were at much lower resolution. The cryoEM density together with X-ray-based coordinates of individual capsid proteins provided the first detailed models of flavi  and alphg 97 VLP  demonstrg 97 VLP . The firg 97 VLP , PRD1  and fitting of X-ray structures of two other capsid gene products  wrote an autobiographical review titled “Confessions of an icosahedral virus crystallographer” . His “coet al. (The virus structures deposited to the PDB over the last 40 years have provided fundamental insights into virus assembly and maturation , the rolet al.  list fouThere can be little doubt that the PDB, together with innovations incorporated from the Virus Particle Explorer database, has played a pivotal role in making icosahedral virus structures accessible for the plethora of studies that have placed many aspects of virology on a structural foundation.The authors declare that they have no conflicts of interest with the contents of this article.