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Nipah RBP DMS

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- + \ No newline at end of file diff --git a/docs/.vitepress/dist/antibody_escape.html b/docs/.vitepress/dist/antibody_escape.html index f1dad890..827e1893 100644 --- a/docs/.vitepress/dist/antibody_escape.html +++ b/docs/.vitepress/dist/antibody_escape.html @@ -8,17 +8,17 @@ - + - - - + + +
Nipah RBP DMS

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Antibody Escape ​

We determined the effects of RBP mutations on neutralization by different RBP-directed antibodies. Antibody selections were performed by incubating pseudovirus libraries with different concentrations of antibody, followed by infection of CHO-bEFNB3 cells to recover barcodes. Neutralization curves were fit on the DMS data with polyclonal.

Individual Antibody Selections ​

Individual antibody selection model fitting notebooks

LibB-230720-m102.4

LibA-230725-m102.4

LibA-231116-m102.4

LibA-230725-nAH1.3

LibB-230630-nAH1.3

LibB-230720-nAH1.3

LibA-231024-HENV26

LibB-230815-HENV26

LibB-230818-HENV26

LibB-230907-HENV26

LibB-230704-HENV32

LibB-230720-HENV32

LibA-230725-HENV32

LibB-230907-HENV32

LibA-230815-HENV103

LibB-230818-HENV103

LibB-230906-HENV103

LibA-230815-HENV117

LibB-230818-HENV117

LibB-230907-HENV117

Average Antibody Escape ​

Averaging antibody escape across libraries and replicate selections.

Average antibody escape notebooks

m102.4

nAH1.3

HENV-26

HENV-32

HENV-103

HENV-117

Antibody Escape Comprehensive Heatmaps ​

Additional control over filtering parameters. Users can adjust different parameters to filter the heatmap data. These provide more information and control compared to the final filtered heatmaps provided on the heatmaps page.

m102.4

HENV-117

HENV-26

HENV-32

HENV-103

nAH1.3

Neutralization Curves ​

Neutralization curves notebook

Neutralization of unmutated Nipah RBP/F pseudovirus by different anti-RBP antibodies.

Antibody Escape Validations ​

Antibody validation notebook

To validate the escape measurements from DMS, we generated single RBP mutant pseudoviruses and tested their neutralization by antibody nAH1.3.

Miscellaneous Figures ​

Escape at Nipah and Hendra polymorphisms and differences

Functional Effect of Antibody Escape Mutations ​

Effects of mutations on cell entry and antibody neutralization

Escape by Site ​

Line plot of average antibody escape at each site

Antibody Escape Analysis Notebook ​

Antibody analysis notebook

Raw Data ​

These data have not been filtered. They are the raw output from dms-vep-pipeline-3. For filtered .csv files, click here.

Individual antibody escape selection files

Averaged effects of RBP mutations on neutralization across replicate selections

antibody m102.4

antibody HENV-117

antibody HENV-26

antibody HENV-32

antibody HENV-103

antibody nAH1.3

Built by Brendan Larsen and Jesse Bloom

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Nipah RBP DMS

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Cell Entry ​

To measure the effects of mutations on RBP-mediated cell entry, we performed 'functional selections' where the frequencies of barcodes were compared between pseudoviruses expressing either Nipah RBP/F or VSV-G. Notebooks below contain information about each step.

Global Epistasis Fitting ​

Individual cell entry selections were fit with multidms to decompose the effects of individual mutations using a global epistasis model.

Individual functional selection global epistasis model fitting notebooks

LibA-230725-CHO-bEFNB3

LibA-230815-CHO-bEFNB3

LibA-230818-CHO-bEFNB3

LibA-230825-CHO-bEFNB3

LibA-230916-CHO-bEFNB3

LibA-231006-CHO-bEFNB3

LibA-231019-CHO-bEFNB3_1

LibA-231019-CHO-bEFNB3_2

LibA-231019-CHO-bEFNB2_1

LibA-231024-CHO-bEFNB3

LibA-231024-CHO-bEFNB2

LibA-231112-CHO-bEFNB3_1

LibA-231112-CHO-bEFNB3_2

LibA-231112-CHO-bEFNB2

LibA-231207-CHO-bEFNB2_1

LibA-231207-CHO-bEFNB2_2

LibA-231207-CHO-bEFNB2_3

LibA-231207-CHO-bEFNB2_4

LibA-231207-CHO-bEFNB2_5

LibA-231222-CHO-bEFNB2

LibB-230630-CHO-C6-nac

LibB-230720-CHO-bEFNB3

LibB-230731-CHO-BA6-nac

LibB-230815-CHO-bEFNB3

LibB-230818-CHO-bEFNB3

LibB-230906-CHO-EFNB3-C6_diffVSV

LibB-230907-CHO-EFNB3-C6-nac_diffVSV

LibB-231105-CHO-EFNB2-BA6-nac_diffVSV

LibB-231108-CHO-EFNB2-BA6-nac_diff_VSV

LibB-231108-CHO-EFNB3-C6-nac_diff_VSV

LibB-231112-CHO-bEFNB2

LibB-231112-CHO-EFNB2-BA6-1

LibB-231112-CHO-EFNB2-BA6-2

LibB-231112-CHO-EFNB3-C6-1

LibB-231112-CHO-EFNB3-C6-2

LibB-231116-CHO-bEFNB3

LibB-231116-CHO-bEFNB2

LibB-231116-CHO-BA6_nac_diff_VSV

LibB-231222-CHO-EFNB2-BA6_diffVSV

LibB-231222-CHO-EFNB2-BA6-nac_diffVSV

Averaging Cell Entry ​

Individual functional selections were averaged between libraries and replicates below.

Notebook averaging the effects of cell entry in CHO-bEFNB2 cells

Notebook averaging the effects of cell entry in CHO-bEFNB3 cells

Comprehensive Cell Entry Heatmaps ​

Additional control over filtering parameters. Users can adjust different parameters to filter the heatmap data. These provide more information and control compared to the final filtered heatmaps provided on the heatmaps page.

CHO-bEFNB2 cell entry heatmap

CHO-bEFNB3 cell entry heatmap

Functional Scores ​

Notebook analyzing the distribution of functional scores for all individual selections.

Functional scores notebook

Analyze Data ​

Notebook analyzing cell entry from filtered data. Make figures for manuscript using python and altair.

Cell entry analysis notebook

Cell Entry Figures ​

TIP

Plots below are interactive. Hover over points to see more information. Click arrow box to view altair plots in separate page.

Cell entry of different RBP regions

Site-averaged Effects of Mutations on Cell Entry ​

Sites in RBP neck and contact sites (ranked from least constrained to most)

Cell Entry Correlations ​

Correlation between site-averaged effects of mutations on cell entry

Correlation between effects of all mutations on cell entry

Cell Entry Validations ​

Cell entry validation notebook

We validated the DMS cell entry measurements by making individual RBP mutants, expressing them on pseudovirus particles, and measuring luciferase following infection.

Raw Data ​

These data have not been filtered. They are the raw output from dms-vep-pipeline-3. For filtered .csv files, click here

Individual cell entry files

Averaged effects of RBP mutations on entry in CHO-bEFNB2 cells across replicates

Averaged effects of RBP mutations on entry in CHO-bEFNB3 cells across replicates

Built by Brendan Larsen and Jesse Bloom

- + \ No newline at end of file diff --git a/docs/.vitepress/dist/hashmap.json b/docs/.vitepress/dist/hashmap.json index a6d3a6aa..26a046fe 100644 --- a/docs/.vitepress/dist/hashmap.json +++ b/docs/.vitepress/dist/hashmap.json @@ -1 +1 @@ -{"index.md":"CQdod3oj","antibody_escape.md":"Boc2s4Bx","interactive.md":"D9-gz2zM","heatmaps.md":"DWOCKQ4i","pipeline_information.md":"DssVjkKV","cell_entry.md":"nyU-7ijj","receptor_binding.md":"CcjtWvHk"} +{"pipeline_information.md":"BGHUoDcX","index.md":"eYsztaDd","interactive.md":"DPEN0iki","heatmaps.md":"DTP-hRMN","antibody_escape.md":"BV6AVDKe","cell_entry.md":"BrmqhwMi","receptor_binding.md":"CQ30tqj1"} diff --git a/docs/.vitepress/dist/heatmaps.html b/docs/.vitepress/dist/heatmaps.html index 7224860b..09a8ebd2 100644 --- a/docs/.vitepress/dist/heatmaps.html +++ b/docs/.vitepress/dist/heatmaps.html @@ -8,17 +8,17 @@ - + - - - + + +
Nipah RBP DMS

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Heatmaps ​

Heatmaps represent one of the best ways to explore deep mutational scanning data. This page contains links to various heatmaps, which show the effects of mutations on three different phenotypes: cell entry, receptor binding, and antibody escape.

TIP

Hover over the heatmaps to see more information about each mutation

Entry Heatmaps ​

Effects of RBP mutations on entry in CHO-bEFNB2 Cells

Effects of RBP mutations on entry in CHO-bEFNB3 Cells

Binding Heatmaps ​

INFO

Mutations with low cell entry scores are masked in dark gray.

bEFNB2 Binding Heatmap

bEFNB3 Binding Heatmap

Antibody Escape Heatmaps ​

INFO

Mutations with low cell entry scores are masked in dark gray. If protein structure is available, distance in angstroms to the closest antibody residue is shown.

m102.4 Antibody Escape

HENV-117 Antibody Escape

HENV-26 Antibody Escape

HENV-103 Antibody Escape

HENV-32 Antibody Escape

nAH1.3 Antibody Escape

Heatmaps of Specific RBP Regions ​

TIP

Click arrow in upper right to view full-sized plots

Effects of mutations on cell entry and binding at receptor contact sites. Receptor contact sites are less constrained for entry in CHO-bEFNB2 cells than CHO-bEFNB3 cells, likely due to ~25-fold higher receptor affinity of RBP to EFNB2 versus EFNB3.

Effects of mutations on cell entry and binding at glycosylation sites ​

Nipah RBP has six sites that are glycosylated. One in the neck (site 159) and five in the head. Here are the effects of mutations on entry and binding.

Effects of mutations on cell entry and binding at polymorphic Nipah sites ​

These sites are polymorphic in Nipah sequences. Most of these sites tolerate multiple mutations.

Effects of mutations on cell entry, organized by type of the unmutated amino acid residue ​

The effects of mutations organized by the unmutated amino acid type. Strong preference for certain amino acids can be seen in certain regions. For example, portions of the stalk only tolerate hydrophobic residues (see sites 101-160 below).

Notebooks ​

Notebook that makes all heatmaps from filtered DMS data. Make figures for manuscript using python and altair.

Heatmap notebook

Built by Brendan Larsen and Jesse Bloom

- + \ No newline at end of file diff --git a/docs/.vitepress/dist/index.html b/docs/.vitepress/dist/index.html index 97c02a8d..e413862e 100644 --- a/docs/.vitepress/dist/index.html +++ b/docs/.vitepress/dist/index.html @@ -8,17 +8,17 @@ - + - - - + + + -
Nipah RBP DMS

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Nipah RBP DMS

Collection of data, figures, and information for the Nipah virus receptor binding protein deep mutational scanning project

Pipeline information and data processing

Analysis of Pacbio and Illumina sequencing data produced from deep mutational scanning experiments

Cell entry

Analysis of functional selections for assessing how RBP mutations impact cell entry

Receptor binding

Analysis of soluble receptor selections for measuring how mutations affect binding to host receptors

Antibody escape

Analysis of antibody neutralization data for finding sites of escape in RBP

Heatmaps

Links to heatmaps showing effects of mutations on cell entry, receptor binding, and antibody escape

Interactive figures

Explore DMS data on Nipah RBP with interactive figures

About ​

This website contains links and information for the Nipah virus receptor binding protein deep mutational scanning project. Look through Jupyter notebooks used in analyses, explore interactive charts, or download raw data. To view more information on the code used to analyze these data and generate the website, click on the GitHub logo in upper right. Interactive charts made with Altair. Embedding of Altair plots was done with custom javascript code from dms-vep. Work done in Bloom Lab, part of Fred Hutch Cancer Center. To access the old version of the homepage built by dms-vep-pipeline-3, click here.

All experiments were performed with non-replicative lentiviral-based pseudoviruses. Ephrin-B2 and Ephrin-B3 orthologs from the bat species Pteropus alecto were used for cell entry and receptor binding assays.

Built by Brendan Larsen and Jesse Bloom

- +
Nipah RBP DMS

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Nipah virus deep mutational scanning

Collection of data, figures, and information for the Nipah virus receptor binding protein deep mutational scanning project

Pipeline information and data processing

Analysis of Pacbio and Illumina sequencing data produced from deep mutational scanning experiments

Cell entry

Analysis of functional selections for assessing how RBP mutations impact cell entry

Receptor binding

Analysis of soluble receptor selections for measuring how mutations affect binding to host receptors

Antibody escape

Analysis of antibody neutralization data for finding sites of escape in the RBP

Heatmaps

Links to heatmaps showing effects of mutations on cell entry, receptor binding, and antibody escape

Interactive figures

Explore DMS data on Nipah RBP with interactive figures

About ​

This website contains links and information for the Nipah virus receptor binding protein deep mutational scanning project. Look through Jupyter notebooks used in analyses, explore interactive charts, or download raw data. To view more information on the code used to analyze these data and generate the website, click on the GitHub logo in upper right. Interactive charts made with Altair. Embedding of Altair plots was done with custom javascript code from dms-vep. All work was done in the Bloom Lab, part of Fred Hutch Cancer Center. To access the old version of the homepage built by dms-vep-pipeline-3, click here.

Scientific Details ​

Deep mutational scanning experiments were performed on the Nipah virus receptor binding protein to measure the effects of mutations on three different phenotypes: cell entry, receptor binding, and antibody escape. We utilized a recently developed lentivirus-based platform to measure the effects of all possible mutations. We used a combination of selections on variant libraries and deep sequencing to measure these effects.

The Nipah virus receptor binding protein is responsible for binding to host cell receptors (ephrin-B2 and -B3) and mediating cell entry. Following receptor binding, RBP triggers a different viral protein, the Fusion (F) protein. Once triggered by RBP, F undergoes irreversible conformational changes to fuse the host and viral membranes. Here are the molecular structures of the RBP and F proteins:

Nipah virus receptor binding protein on left, fusion protein on right. Colors are individual monomers.

Biosafety ​

All experiments were performed with non-replicative lentiviral-based pseudoviruses in a biosafety-level 2 laboratory by trained personel. Rescue of lentiviruses from cells requires co-transfection with multiple different plasmids, limiting the risk of recombination. Ephrin-B2 and ephrin-B3 orthologs from the bat species Pteropus alecto were used for cell entry and receptor binding assays to limit information hazards of identifying human-specific adaptive mutations.

Built by Brendan Larsen and Jesse Bloom

+ \ No newline at end of file diff --git a/docs/.vitepress/dist/interactive.html b/docs/.vitepress/dist/interactive.html index 06ae5416..90b36555 100644 --- a/docs/.vitepress/dist/interactive.html +++ b/docs/.vitepress/dist/interactive.html @@ -8,17 +8,17 @@ - + - - - + + + -
Nipah RBP DMS

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Interactive Figures ​

Explore Nipah virus RBP deep mutational scanning data with interactive charts.

To explore heatmaps, click here

TIP

Click white square in the upper right of each plot to view full-sized versions.

Cell Entry ​


Correlations ​

Correlations by Site ​


Notebooks ​

Link to notebooks showing how interactive figures were made:

Interactive figures notebook

Built by Brendan Larsen and Jesse Bloom

- +
Nipah RBP DMS

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Interactive Figures ​

Explore Nipah virus RBP deep mutational scanning data with interactive charts.

To explore heatmaps

TIP

Click white square in the upper right of each plot to view full-sized versions.

Cell Entry ​


Correlations ​

Correlations by Site ​


Notebooks ​

Link to notebooks showing how interactive figures were made:

Interactive figures notebook

Built by Brendan Larsen and Jesse Bloom

+ \ No newline at end of file diff --git a/docs/.vitepress/dist/pipeline_information.html b/docs/.vitepress/dist/pipeline_information.html index 035a8149..ef724e82 100644 --- a/docs/.vitepress/dist/pipeline_information.html +++ b/docs/.vitepress/dist/pipeline_information.html @@ -8,11 +8,11 @@ - + - - - + + + @@ -20,7 +20,7 @@
Nipah RBP DMS

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Pipeline Information ​

To link barcodes to specific variants present in each RBP sequence, we performed Pacbio sequencing on variant libraries. Full-length RBP consensus sequences were made to determine which mutations were present in each library. DMS data were analyzed with dms-vep-pipeline-3. This pipeline utilizes the alignparse package. The general steps are listed below.

Build Pacbio Sequences ​

PacBio consensus sequences notebook

Builds the Pacbio consensus sequence. Parameters used are:

max_minor_sub_frac=0.2
 max_minor_indel_frac=0.2
 min_support=3

These parameters filter consensus sequences generated from Pacbio CCS sequencing and assembly. If an assembled RBP sequence has a mutation or indel in more than 20% of the reads, it will be discarded. Consensus sequences must have at least three reads to be included as variants.

With alignparse, reads were mapped to a reference sequence, and clipped based on parameters in this config file.

Analyze PacBio CCS Reads ​

Analyze Pacbio CCS reads notebook

Reports information about CCS read filtering.

Build Codon Variants Notebook ​

Build codon variants notebook

Builds the codon-variant table from PacBio consensus sequences that links barcodes and RBP mutations.

Link to codon-variant table .csv file

Illumina Variant Counts ​

Once the barcodes are linked to mutations in the codon-variant table, all sequencing data is generated with Illumina on a small sequence fragment to obtain the relative frequencies of barcodes in each selection experiment. The config file linked below specifies the parameters used for converting barcode counts to functional scores, which are used to estimate cell entry.

Analysis of variant counts notebook

Link to raw barcode count .csv files

Link to functional selection config file

Filtering Selection Data ​

Filtering notebook

The final cell entry, receptor binding, and antibody escape data were filtered based on parameters that are contained within the nipah_config.yaml file. More information about these parameters are listed in the notebook.

Filtered Data ​

These data have been filtered and are the best choice for anyone interested in analyzing the data themselves. For unfiltered raw .csv files of mutational effects on different phenotypes, go to individual pages to view and download.

Cell Entry ​

CHO-bEFNB2 entry filtered (.csv)

CHO-bEFNB3 entry filtered (.csv)

Receptor Binding ​

bEFNB2 monomeric binding filtered (.csv)

bEFNB3 dimeric binding filtered (.csv)

Antibody Escape ​

Antibody escape filtered (.csv)

Miscellaneous Notebooks ​

Notebook for finding correlations between libraries and making histogram of variants

Notebook for making a Nipah phylogeny

Notebook for making specific file formats (.defattr), to map site-averaged scores onto protein structures in Chimera

Notebook for calculating atomic distances between residues from a PDB file

Notebook for finding variable sites in Nipah or Henipavirus alignments

Built by Brendan Larsen and Jesse Bloom

- + \ No newline at end of file diff --git a/docs/.vitepress/dist/receptor_binding.html b/docs/.vitepress/dist/receptor_binding.html index 9a8e7f53..13ed6031 100644 --- a/docs/.vitepress/dist/receptor_binding.html +++ b/docs/.vitepress/dist/receptor_binding.html @@ -8,17 +8,17 @@ - + - - - + + +
Nipah RBP DMS

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Receptor Binding ​

To understand how mutations affect binding to ephrin receptors, we performed selections on our pseudovirus libraries with soluble bat EFNB2 and EFNB3. Neutralization of pseudovirus variants serves as a proxy for receptor binding. Neutralization curves were fit with polyclonal.

Individual Receptor Binding Selections ​

Individual antibody selection model fitting notebooks

LibB-231108-bEFNB2-monomeric

LibA-231112-bEFNB2-monomeric

LibA-231207-bEFNB2-monomeric

LibA-231222-bEFNB2-monomeric

LibB-231222-bEFNB2-monomeric

LibA-230818-EFNB3-dimeric

LibA-230825-bEFNB3-dimeric

LibB-230907-bEFNB3-dimeric

Average Receptor Binding ​

These notebooks average effects of mutations on receptor binding across libraries and replicate selections.

bEFNB2-monomeric

bEFNB3-dimeric

Comprehensive Receptor Binding Heatmaps ​

Additional control over filtering parameters. Users can adjust different parameters to filter the heatmap data. These provide more information and control compared to the final filtered heatmaps provided on the heatmaps page.

bEFNB2-monomeric heatmap

bEFNB3-dimeric heatmap

Neutralization of Nipah pseudoviruses ​

Pseudoviruses expressing Nipah virus RBP/F are neutralized by soluble bat ephrins.

Ephrin neutralization of pseudoviruses expressing unmutated Nipah RBP/F

Binding Correlations ​

Effects of mutations on binding to bEFNB2 and bEFNB3, with mutations of interest highlighted

Interactive plot of bEFNB2 and bEFNB3 site-averaged binding correlations

Binding Validations ​

Binding Validation by BLI ​

BLI validations notebook

Correlation of biolayer interferometry affinity measurements with DMS

Binding Validation by Neutralization ​

Binding validations notebook

Neutralization of single RBP mutant pseudoviruses and correlation with DMS

Analysis Notebooks ​

Notebook analyzing receptor binding from filtered data. Make figures for manuscript using python and altair.

Binding analysis notebook

Raw Data ​

These data have not been filtered. They are the raw output from dms-vep-pipeline-3. For filtered .csv files, click here

Individual receptor binding selection files

Averaged effects of RBP mutations on bEFNB2 binding across replicates

Averaged effects of RBP mutations on bEFNB3 binding across replicates

Built by Brendan Larsen and Jesse Bloom

- + \ No newline at end of file diff --git a/docs/antibody_escape.md b/docs/antibody_escape.md index c6093a47..a1046ed0 100755 --- a/docs/antibody_escape.md +++ b/docs/antibody_escape.md @@ -1,6 +1,6 @@ -# Antibody Escape +# Antibody Escape -We determined the effects of RBP mutations on neutralization by different RBP-directed antibodies. Antibody selections were performed by incubating pseudovirus libraries with different concentrations of antibody, followed by infection of CHO-bEFNB3 cells to recover barcodes. Neutralization curves were fit on the DMS data with [`polyclonal`](https://github.com/jbloomlab/polyclonal){target="_self"}. +We determined the effects of RBP mutations on neutralization by different RBP-directed antibodies. Antibody selections were performed by incubating pseudovirus libraries with different concentrations of antibody, followed by infection of CHO-bEFNB3 cells to recover barcodes. Neutralization curves were fit on the DMS data with [`polyclonal`](https://github.com/jbloomlab/polyclonal){target="_self"}. ## Individual Antibody Selections ::: details Individual antibody selection model fitting notebooks diff --git a/docs/index.md b/docs/index.md index 5234170b..9e710479 100755 --- a/docs/index.md +++ b/docs/index.md @@ -3,42 +3,59 @@ layout: home hero: - name: "Nipah RBP DMS" + name: "Nipah virus deep mutational scanning" tagline: "Collection of data, figures, and information for the Nipah virus receptor binding protein deep mutational scanning project" image: /images/entry_tetramer.png features: - title: Pipeline information and data processing - details: Analysis of Pacbio and Illumina sequencing data produced from deep mutational scanning experiments + details: Analysis of Pacbio and Illumina sequencing data produced from deep mutational scanning experiments link: /pipeline_information - + - title: Cell entry - details: Analysis of functional selections for assessing how RBP mutations impact cell entry + details: Analysis of functional selections for assessing how RBP mutations impact cell entry link: /cell_entry - title: Receptor binding - details: Analysis of soluble receptor selections for measuring how mutations affect binding to host receptors + details: Analysis of soluble receptor selections for measuring how mutations affect binding to host receptors link: /receptor_binding - title: Antibody escape - details: Analysis of antibody neutralization data for finding sites of escape in RBP + details: Analysis of antibody neutralization data for finding sites of escape in the RBP link: /antibody_escape - title: Heatmaps - details: Links to heatmaps showing effects of mutations on cell entry, receptor binding, and antibody escape + details: Links to heatmaps showing effects of mutations on cell entry, receptor binding, and antibody escape link: /heatmaps - title: Interactive figures - details: Explore DMS data on Nipah RBP with interactive figures + details: Explore DMS data on Nipah RBP with interactive figures link: /interactive --- ### About -This website contains links and information for the Nipah virus receptor binding protein deep mutational scanning project. Look through Jupyter notebooks used in analyses, explore [interactive charts](/interactive), or download raw [data](/pipeline_information#filtered-data). To view more information on the code used to analyze these data and generate the website, click on the GitHub logo in upper right. Interactive charts made with [Altair](https://altair-viz.github.io){target="_self"}. Embedding of Altair plots was done with custom javascript code from [dms-vep](https://github.com/dms-vep/dms-vep.github.io){target="_self"}. Work done in [Bloom Lab](https://research.fredhutch.org/bloom/en.html){target="_self"}, part of [Fred Hutch Cancer Center](https://www.fredhutch.org/en.html){target="_self"}. To access the old version of the homepage built by `dms-vep-pipeline-3`, click [here](/index){target='_self'}. +This website contains links and information for the Nipah virus receptor binding protein deep mutational scanning project. Look through Jupyter notebooks used in analyses, explore [interactive charts](/interactive), or download raw [data](/pipeline_information#filtered-data). To view more information on the code used to analyze these data and generate the website, click on the GitHub logo in upper right. Interactive charts made with [Altair](https://altair-viz.github.io){target="_self"}. Embedding of Altair plots was done with custom javascript code from [dms-vep](https://github.com/dms-vep/dms-vep.github.io){target="_self"}. All work was done in the [Bloom Lab](https://research.fredhutch.org/bloom/en.html){target="_self"}, part of [Fred Hutch Cancer Center](https://www.fredhutch.org/en.html){target="_self"}. To access the old version of the homepage built by `dms-vep-pipeline-3`, click [here](/index){target='_self'}. + +### Scientific Details + +Deep mutational scanning experiments were performed on the Nipah virus receptor binding protein to measure the effects of mutations on three different phenotypes: cell entry, receptor binding, and antibody escape. We utilized a [recently developed lentivirus-based platform](https://pubmed.ncbi.nlm.nih.gov/36868218/){target='_self'} to measure the effects of all possible mutations. We used a combination of selections on variant libraries and deep sequencing to measure these effects. + +The Nipah virus receptor binding protein is responsible for binding to host cell receptors (ephrin-B2 and -B3) and mediating cell entry. Following receptor binding, RBP triggers a different viral protein, the Fusion (F) protein. Once triggered by RBP, F undergoes irreversible conformational changes to fuse the host and viral membranes. Here are the molecular structures of the RBP and F proteins: + +
+ +
+ +
Nipah virus receptor binding protein on left, fusion protein on right. Colors are individual monomers.
+ +### Biosafety -All experiments were performed with non-replicative lentiviral-based pseudoviruses. Ephrin-B2 and Ephrin-B3 orthologs from the bat species *Pteropus alecto* were used for cell entry and receptor binding assays. +All experiments were performed with non-replicative lentiviral-based pseudoviruses in a biosafety-level 2 laboratory by trained personel. Rescue of lentiviruses from cells requires co-transfection with multiple different plasmids, limiting the risk of recombination. Ephrin-B2 and ephrin-B3 orthologs from the bat species *Pteropus alecto* were used for cell entry and receptor binding assays to limit information hazards of identifying human-specific adaptive mutations. diff --git a/docs/interactive.md b/docs/interactive.md index f577f242..1b8217dd 100755 --- a/docs/interactive.md +++ b/docs/interactive.md @@ -2,7 +2,7 @@ Explore Nipah virus RBP deep mutational scanning data with interactive charts. -To explore heatmaps, click [here](/heatmaps) +To explore [heatmaps](/heatmaps) ::: tip