Multimodal HLA-I genotype regulation by human cytomegalovirus US10 and resulting surface patterning
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eLife assessment
This study presents a useful finding on a virally encoded immune-evasin which differentially inhibits antigen presentation by cellular protein complexes called Major histocompatibility complex (MHC) class I, thereby diminishing the activation of cytotoxic T cells. The evidence supporting the claims of the authors is solid, although the addition of more mechanistic insights would strengthen the study. The work will be of interest to virologists and immunologists working on the adaptive immune response to herpesviral infection. Some conclusions would require additional experimental support.
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Abstract
Human leucocyte antigen class I (HLA-I) molecules play a central role for both NK and T-cell responses that prevent serious human cytomegalovirus (HCMV) disease. To create opportunities for viral spread, several HCMV-encoded immunoevasins employ diverse strategies to target HLA-I. Among these, the glycoprotein US10 is so far insufficiently studied. While it was reported that US10 interferes with HLA-G expression, its ability to manipulate classical HLA-I antigen presentation remains unknown. In this study, we demonstrate that US10 recognizes and binds to all HLA-I (HLA-A, -B, -C, -E, -G) heavy chains. Additionally, impaired recruitment of HLA-I to the peptide loading complex was observed. Notably, the associated effects varied significantly dependending on HLA-I genotype and allotype: (i) HLA-A molecules evaded downregulation by US10, (ii) tapasin-dependent HLA-B molecules showed impaired maturation and cell surface expression, and (iii) β 2 m-assembled HLA-C, in particular HLA-C*05:01 and -C*12:03, and HLA-G were strongly retained in complex with US10 in the endoplasmic reticulum. These genotype-specific effects on HLA-I were confirmed through unbiased HLA-I ligandome analyses. Furthermore, in HCMV-infected fibroblasts inhibition of overlapping US10 and US11 transcription had little effect on HLA-A, but induced HLA-B antigen presentation. Thus, the US10-mediated impact on HLA-I results in multiple geno- and allotypic effects in a so far unparalleled and multimodal manner.
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Author response:
Reviewer #2 (Public Review):
The manuscript entitled " Multimodal HLA-I genotypes regulation by human cytomegalovirus US10 and resulting surface patterning" by Gerke et al describes the biochemical analysis of US10-mediated down regulation of HLA-I molecules. The authors systemically examine the surface expression of different HLA-I alleles in cells expressing US10 and interactions of US10 with HLA-I and antigen presentation machinery. Further, studies examined genotypic and allotypic differences during expression of US10/US11 transcripts suggest a different allelic class I downregulation. In general, the authors have included data supporting the major claims. Yet, the conclusions and findings of the study only marginally advance the overall understanding of HCMV viral evasion and the mechanism of US10 function.
Str…
Author response:
Reviewer #2 (Public Review):
The manuscript entitled " Multimodal HLA-I genotypes regulation by human cytomegalovirus US10 and resulting surface patterning" by Gerke et al describes the biochemical analysis of US10-mediated down regulation of HLA-I molecules. The authors systemically examine the surface expression of different HLA-I alleles in cells expressing US10 and interactions of US10 with HLA-I and antigen presentation machinery. Further, studies examined genotypic and allotypic differences during expression of US10/US11 transcripts suggest a different allelic class I downregulation. In general, the authors have included data supporting the major claims. Yet, the conclusions and findings of the study only marginally advance the overall understanding of HCMV viral evasion and the mechanism of US10 function.
Strengths:
The studies are well characterized and the studies utilize diverse HLA-I and HCMV viral molecules. The biochemistry is excellent and is of high quality. Importantly, the study describes HLA-I allelic specific HCMV down regulation at the cell surface and molecular levels.
Weaknesses:
(1) The authors use over expressive language such as "strong binding" that does not have a quantitative value and it is relative to the specific assay with only small differences among the factors.
We have changed the language to avoid non-quantitative expressions.
(2) The US10 binding to the HLA-I did not correlate with class I surface levels suggesting that binding to the APC machinery (Figure 1); hence, why does the binding of US10 to the APC define its mechanism of action.
We hypothesized that since binding to HLA-I allomorphs did not correlate with surface expression, further factors could be involved in regulation. Since the PLC (APC machinery) plays a major role for HLA-I expression, it was relevant to investigate this. The new data underlines the importance of the PLC for US10-mediated HLA-I regulation.
(3) The innovative and significant aspects of the study are limited. The study does not delineate the US10 mechanism of action or show data in which US10-mediated MHC class I down regulation impacts adaptive or innate immune function.
These remarks are important. We want to emphasize the variable impact of US10 on HLA-I. To our knowledge previous studies have not uncovered genotype-dependent effects on HLA-I as distinct as those observed with US10, indicating that US10 may exploit aspects of HLA-I that are yet to be fully elucidated. Therefore, confirming these findings is crucial for our study. The quantitative analysis of the HeLa HLA-I ligandome in US10-expressing cells strongly supports this conclusion. The precise quantification of HLA-I peptide ligands was made possible through collaboration with Dr. Andreas Schlosser from Würzburg, Germany, who possesses profound expertise in this specific method. Thus, in our opinion, this revision has enabled us to advance innovation and, importantly, enhance the significance of our study.
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eLife assessment
This study presents a useful finding on a virally encoded immune-evasin which differentially inhibits antigen presentation by cellular protein complexes called Major histocompatibility complex (MHC) class I, thereby diminishing the activation of cytotoxic T cells. The evidence supporting the claims of the authors is solid, although the addition of more mechanistic insights would strengthen the study. The work will be of interest to virologists and immunologists working on the adaptive immune response to herpesviral infection. Some conclusions would require additional experimental support.
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Reviewer #1 (Public Review):
HMCV encodes various immunoevasins to inhibit being presented by MHC class I molecules to the cytotoxic cells of the immune system. Here, the authors studied the role and specificity of US10, a relatively uncharacterized immunoevasin from HCMV. They found that US10 differentially affects antigen presentation by different MHC class I allotypes. HLA-A and certain HLA-B and C alleles (so-called "tapasin-independent") were unaffected, while other HLA-B and C alleles (so-called "tapasin-dependent") as well as HLA-G were negatively affected. US10 can bind to different MHC class I allotypes, which inhibits their incorporation into peptide loading complex and slowers maturation. By comparing US10 to the other well-studied immunoevasins from HCMV, US2, US3, and US11, the authors demonstrated only partial overlap …
Reviewer #1 (Public Review):
HMCV encodes various immunoevasins to inhibit being presented by MHC class I molecules to the cytotoxic cells of the immune system. Here, the authors studied the role and specificity of US10, a relatively uncharacterized immunoevasin from HCMV. They found that US10 differentially affects antigen presentation by different MHC class I allotypes. HLA-A and certain HLA-B and C alleles (so-called "tapasin-independent") were unaffected, while other HLA-B and C alleles (so-called "tapasin-dependent") as well as HLA-G were negatively affected. US10 can bind to different MHC class I allotypes, which inhibits their incorporation into peptide loading complex and slowers maturation. By comparing US10 to the other well-studied immunoevasins from HCMV, US2, US3, and US11, the authors demonstrated only partial overlap between them suggesting the cumulative action of immunoevasins in inhibiting MHC class I antigen presentation of HMCV epitopes. This work contributes to our understanding of the complex immune evasion mechanism by HCMV.
The strengths include using a broad use of available techniques, including overexpression of US10 and US10 siRNA in the infection context that allowed comparison of its net and cumulative effects. Bioinformatic analysis of US10 and US11 to describe how transcription and expression of these two gene products contribute to the control of immunoevasion by HCMV. The conclusions are mostly supported by the experiments.
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Reviewer #2 (Public Review):
The manuscript entitled " Multimodal HLA-I genotypes regulation by human cytomegalovirus US10 and resulting surface patterning" by Gerke et al describes the biochemical analysis of US10-mediated down regulation of HLA-I molecules. The authors systemically examine the surface expression of different HLA-I alleles in cells expressing US10 and interactions of US10 with HLA-I and antigen presentation machinery. Further, studies examined genotypic and allotypic differences during expression of US10/US11 transcripts suggest a different allelic class I downregulation. In general, the authors have included data supporting the major claims. Yet, the conclusions and findings of the study only marginally advance the overall understanding of HCMV viral evasion and the mechanism of US10 function.
Strengths:
The studies …Reviewer #2 (Public Review):
The manuscript entitled " Multimodal HLA-I genotypes regulation by human cytomegalovirus US10 and resulting surface patterning" by Gerke et al describes the biochemical analysis of US10-mediated down regulation of HLA-I molecules. The authors systemically examine the surface expression of different HLA-I alleles in cells expressing US10 and interactions of US10 with HLA-I and antigen presentation machinery. Further, studies examined genotypic and allotypic differences during expression of US10/US11 transcripts suggest a different allelic class I downregulation. In general, the authors have included data supporting the major claims. Yet, the conclusions and findings of the study only marginally advance the overall understanding of HCMV viral evasion and the mechanism of US10 function.
Strengths:
The studies are well characterized and the studies utilize diverse HLA-I and HCMV viral molecules. The biochemistry is excellent and is of high quality. Importantly, the study describes HLA-I allelic specific HCMV down regulation at the cell surface and molecular levels.Weaknesses:
(1) The authors use over expressive language such as "strong binding" that does not have a quantitative value and it is relative to the specific assay with only small differences among the factors.
(2) The US10 binding to the HLA-I did not correlate with class I surface levels suggesting that binding to the APC machinery (Figure 1); hence, why does the binding of US10 to the APC define its mechanism of action.
(3) The innovative and significant aspects of the study are limited. The study does not delineate the US10 mechanism of action or show data in which US10-mediated MHC class I down regulation impacts adaptive or innate immune function. -
Reviewer #3 (Public Review):
Correlation of the HLA-B effects with previously demonstrated allelic differences in dependence on the peptide loading complex (PLC) component chaperone/editor tapasin and demonstration that US10 does not bind the PLC reflect on possible mechanisms of US10 function. Thus, this paper adds new information that may be integrated into evolving models of the steps of MHC-I dependent antigen presentation and how viruses counter immune recognition for their own benefit. Clearer focus on the proposed models for the function of US10 and its mechanism--i.e. what experiments address the mechanism and what additional finding might clarify the mechanism would be helpful.
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