Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail
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{"title"=>"Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail", "type"=>"journal", "authors"=>[{"first_name"=>"Walter", "last_name"=>"Muranyi", "scopus_author_id"=>"6603637980"}, {"first_name"=>"Sebastian", "last_name"=>"Malkusch", "scopus_author_id"=>"37113717200"}, {"first_name"=>"Barbara", "last_name"=>"Müller", "scopus_author_id"=>"7403187010"}, {"first_name"=>"Mike", "last_name"=>"Heilemann", "scopus_author_id"=>"6701502015"}, {"first_name"=>"Hans Georg", "last_name"=>"Kräusslich", "scopus_author_id"=>"7006112877"}], "year"=>2013, "source"=>"PLoS Pathogens", "identifiers"=>{"pui"=>"369459545", "issn"=>"15537366", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "doi"=>"10.1371/journal.ppat.1003198", "scopus"=>"2-s2.0-84878279566", "pmid"=>"23468635", "sgr"=>"84878279566"}, "id"=>"c8f554b1-b869-39bc-9b9e-6928b75e4b6a", "abstract"=>"The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.", "link"=>"http://www.mendeley.com/research/superresolution-microscopy-reveals-specific-recruitment-hiv1-envelope-proteins-viral-assembly-sites", "reader_count"=>93, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>7, "Researcher"=>28, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>27, "Student > Postgraduate"=>6, "Student > Master"=>10, "Other"=>3, "Student > Bachelor"=>4, "Professor"=>3}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>7, "Researcher"=>28, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>27, "Student > Postgraduate"=>6, "Student > Master"=>10, "Other"=>3, "Student > Bachelor"=>4, "Professor"=>3}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Engineering"=>6, "Biochemistry, Genetics and Molecular Biology"=>14, "Agricultural and Biological Sciences"=>46, "Medicine and Dentistry"=>3, "Neuroscience"=>1, "Physics and Astronomy"=>12, "Chemistry"=>4, "Immunology and Microbiology"=>4}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>6}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>4}, "Physics and Astronomy"=>{"Physics and Astronomy"=>12}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>46}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>14}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"Czech Republic"=>1, "United States"=>2, "United Kingdom"=>1, "France"=>1, "Switzerland"=>1, "Germany"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/974471"], "description"=>"<p>HeLa cells were transfected with equimolar amounts of pCHIV and pCHIV<sup>mEosFP</sup>. At 24 hpt, cells were fixed and HIV-1 assembly sites were imaged by PALM as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4\" target=\"_blank\">Materials and Methods</a>. Summed intensity TIRF (<b>A</b>) and high resolution PALM (<b>B</b>) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. The boxed region in (B) corresponds to the region of interest shown as an enlarged high resolution image in (<b>C</b>). Scale bar in (C) corresponds to 1 µm. (<b>D</b>) Four individual HIV-1 assembly sites from the boxed regions shown in (C). Scale bar corresponds to 100 nm.</p>", "links"=>[], "tags"=>["plasma", "membrane", "hela", "cells", "imaged", "super-resolution", "tirf"], "article_id"=>642485, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_Gag_mEosFP_at_the_plasma_membrane_of_HeLa_cells_imaged_by_super_resolution_TIRF_microscopy_/642485", "title"=>"Distribution of Gag.mEosFP at the plasma membrane of HeLa cells imaged by super-resolution TIRF microscopy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:02:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/974474"], "description"=>"<p>HeLa cells were transfected with equimolar amounts of pCHIV and pCHIV<sup>mEosFP</sup>. Cells were fixed 24 hpt, stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647 and imaged by dSTORM as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4\" target=\"_blank\">Materials and Methods</a>. Summed intensity TIRF (<b>A</b>) and high resolution dSTORM (<b>B</b>) microscopy images of a representative cell are displayed. Scale bars correspond to 2 µm. (<b>C</b>) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. HeLa cells transfected with equimolar amounts of pCHIV and pCHIV<sup>mEosFP</sup> were fixed and stained by indirect immunofluorescence using MAb 2G12 and goat anti-human Alexa Fluor 647. Cells were subjected to dual-color super-resolution microscopy as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4\" target=\"_blank\">Materials and Methods</a>. Scale bar corresponds to 1 µm. (<b>D</b>) Individual HIV-1 assembly sites from the boxed regions are shown. Scale bar corresponds to 100 nm. (<b>E</b>) Env surface distribution on extracellular immature HIV-1 particles. Particles were purified from the supernatant of 293T cells transfected with pCHIV carrying a point mutation in the HIV-1 protease active site <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#ppat.1003198-Chojnacki1\" target=\"_blank\">[39]</a>. An eGFP-tagged version of the accessory protein Vpr <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#ppat.1003198-McDonald1\" target=\"_blank\">[63]</a> was included as a marker to localize the position of individual virions in diffraction limited images. Virions were adhered to fibronectin-coated coverslips, fixed and immunostained with MAb 2G12 and goat anti-human Alexa Fluor 647. The panel shows images of four individual particles with the eGFP signal (green) recorded in diffraction limited mode and the Alexa Fluor 647 (red) signal recorded by dSTORM. Scale bars correspond to 100 nm. (<b>F</b>) Comparison of the Env signal intensities on single extracellular immature particles with the Env signal intensities colocalizing with HIV-1 assembly sites defined by the dSTORM signal for Gag.mEosFP. Fluorescence intensities of the Alexa Fluor 647 signals were determined for the super-resolution images of ten individual particles each. The histogram shows the average values and SD of the fluorescence intensity.</p>", "links"=>[], "tags"=>["hiv-1", "env", "plasma", "membrane", "hela", "cells", "visualized", "super-resolution", "tirf"], "article_id"=>642486, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_HIV_1_Env_at_the_plasma_membrane_of_HeLa_cells_visualized_by_super_resolution_TIRF_microscopy_/642486", "title"=>"Distribution of HIV-1 Env at the plasma membrane of HeLa cells visualized by super-resolution TIRF microscopy.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:02:35"}
  • {"files"=>["https://ndownloader.figshare.com/files/974475"], "description"=>"<p>(<b>A, B</b>) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV and pCHIV<sup>mEosFP</sup>. Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4\" target=\"_blank\">Materials and Methods</a>. (<b>A</b>) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (<b>B</b>) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (<b>C, D</b>) Env distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV and pCHIV<sup>mEosFP</sup> (<b>C</b>) or with pEnv(wt) (<b>D</b>), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dual-color super-resolution microscopy as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4\" target=\"_blank\">Materials and Methods</a>. Scale bars represent 1 µm.</p>", "links"=>[], "tags"=>["viral"], "article_id"=>642487, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparative_analysis_of_Env_wt_distribution_expressed_in_the_viral_context_or_alone_/642487", "title"=>"Comparative analysis of Env(wt) distribution expressed in the viral context or alone.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:02:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/974478"], "description"=>"<p>(<b>A, B</b>) Distribution of HIV-1 Gag and Env at the plasma membrane of HeLa cells transfected with equimolar amounts of pCHIV.Env(ΔCT) and pCHIV<sup>mEosFP</sup>. Env(ΔCT). Unfixed cells were stained by indirect immunofluorescence using Fab 2G12 and Fab goat anti-human Alexa Fluor 647, fixed, and subjected to dual-color super-resolution microscopy as described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#s4\" target=\"_blank\">Materials and Methods</a>. (<b>A</b>) Region from the plasma membrane of a representative cell, showing the superposition of a PALM image for Gag.mEosFP (green) and the corresponding dSTORM image of Env(ΔCT) stained with Alexa Fluor 647 (red), respectively. Scale bar corresponds to 1 µm. (<b>B</b>) Enlargement of three individual assembly sites from the boxed regions indicated in (A). The figure shows merged super-resolution images (left panels), the dSTORM Env Alexa Fluor 647 image (middle panels) and individual Alexa Fluor 647 localizations from all images recorded in the defined area as black dots, with a black circle representing the rims of the Gag cluster (right panels), respectively. Scale bar corresponds to 100 nm. (<b>C, D</b>) Comparison of Env(ΔCT) distribution patterns in the presence (C) and absence (D) of other HIV-1 derived proteins. HeLa cells were transfected with equimolar amounts of pCHIV.Env(ΔCT) and pCHIV<sup>mEosFP</sup>Env(ΔCT) (<b>C</b>) or with pEnv(ΔCT) (<b>D</b>), respectively. Unfixed cells were stained with 2G12 Fab and Alexa Fluor 647 Fab, fixed, and visualized by dSTORM as described. Scale bars represent 1 µm.</p>", "links"=>[], "tags"=>["viral"], "article_id"=>642490, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparative_analysis_of_Env_916_CT_distribution_expressed_in_the_viral_context_or_alone_/642490", "title"=>"Comparative analysis of Env(ΔCT) distribution expressed in the viral context or alone.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:03:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/974479"], "description"=>"<p>HeLa cells were transfected with proviral constructs carrying both wt Gag and wt Env (<b>A</b>), wt Gag and Env(ΔCT) (<b>B</b>), Gag carrying the MA mutation and wt Env (<b>C</b>), or comprising both mutated MA and Env(ΔCT) (<b>D</b>), respectively. Fixed cells were stained by indirect immunofluorescence using MAb APR342 and goat anti-mouse Alexa Fluor 532 and MAb 2G12 and goat anti-human Alexa Fluor 647, and subjected to dual-color dSTORM. Scale bar corresponds to 1 µm. An overview of respective cells is presented in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003198#ppat.1003198.s007\" target=\"_blank\">Figure S7</a>.</p>", "links"=>[], "tags"=>["env-interaction", "deficient", "gag"], "article_id"=>642491, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_Env_wt_or_Env_916_CT_in_the_context_of_an_Env_interaction_deficient_Gag_variant_/642491", "title"=>"Distribution of Env(wt) or Env(ΔCT) in the context of an Env-interaction deficient Gag variant.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:03:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/974480"], "description"=>"<p>Global cluster analysis of Env distribution at the plasma membrane was performed based on super-resolution images of HeLa cells transfected with pCHIV/pCHIV<sup>mEos.FP</sup>, pCHIV. Env(ΔCT)/pCHIV<sup>mEosFP</sup>Env(ΔCT), pEnv(wt) or pEnv(ΔCT), or of A3.01 T-cells nucleofected with pCHIV/pCHIV<sup>mEos.FP</sup> or pCHIV.Env(ΔCT)/pCHIV<sup>mEosFP</sup>Env(ΔCT), respectively. (<b>A</b>) Image-based, morphological cluster analysis of entire cells. (<b>B</b>) Differential distribution of cluster size for Env(wt) on the surface of HeLa cells in the presence and absence of other viral proteins. The curve was derived by subtraction of the normalized distribution of cluster size obtained by image-based cluster analysis for Env(wt) expressed in the HIV-1 context from that obtained upon expression of Env(wt) alone. (<b>C</b>) Coordinate based all-distance distribution analysis by Ripley's H-function. Statistical evaluation of the maximal H-values [nm] was performed as a correlate of the average cluster diameter. (<b>D</b>) Statistical evaluation of the amplitude of the H-function [a.u.] as a measure of the average degree of clustering. Box plots display 5<sup>th</sup> percentile, 25<sup>th</sup> percentile, median (straight line), mean (square), 75<sup>th</sup> percentile and 95<sup>th</sup> percentile.</p>", "links"=>[], "tags"=>["hiv-1", "env", "membrane"], "article_id"=>642493, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Computational_cluster_analysis_of_HIV_1_Env_membrane_distribution_/642493", "title"=>"Computational cluster analysis of HIV-1 Env membrane distribution.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:03:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/974483"], "description"=>"<p>Image-based, averaged assembly site analysis performed on super-imposed high-resolution images of individual Gag.mEosFP clusters (green) from cells transfected with pCHIV/pCHIV<sup>mEosFP</sup> (<b>A</b>) or pCHIV.Env(ΔCT)/pCHIV<sup>mEosFP</sup>Env(ΔCT) (<b>B</b>), respectively (n = 7 assembly sites per condition) and the averaged Env Alexa Fluor 647 signal in the respective area (red). Graphs show the profile of intensity through the center of the averaged, overlaid intensity images: green line, Gag.mEosFP; red line, Env. Scale bars represent 50 nm.</p>", "links"=>[], "tags"=>["super-resolution", "env", "hiv-1"], "article_id"=>642495, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Averaged_super_resolution_intensity_distribution_of_Env_at_HIV_1_assembly_sites_/642495", "title"=>"Averaged super-resolution intensity distribution of Env at HIV-1 assembly sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-03-01 14:03:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/974489", "https://ndownloader.figshare.com/files/974490", "https://ndownloader.figshare.com/files/974491", "https://ndownloader.figshare.com/files/974492", "https://ndownloader.figshare.com/files/974493", "https://ndownloader.figshare.com/files/974494", "https://ndownloader.figshare.com/files/974495", "https://ndownloader.figshare.com/files/974496"], "description"=>"<div><p>The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread <i>in vivo</i>. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.</p> </div>", "links"=>[], "tags"=>["super-resolution", "microscopy", "reveals", "recruitment", "hiv-1", "proteins", "viral", "sites", "c-terminal", "tail"], "article_id"=>642500, "categories"=>["Virology"], "users"=>["Walter Muranyi", "Sebastian Malkusch", "Barbara Müller", "Mike Heilemann", "Hans-Georg Kräusslich"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003198.s001", "https://dx.doi.org/10.1371/journal.ppat.1003198.s002", "https://dx.doi.org/10.1371/journal.ppat.1003198.s003", "https://dx.doi.org/10.1371/journal.ppat.1003198.s004", "https://dx.doi.org/10.1371/journal.ppat.1003198.s005", "https://dx.doi.org/10.1371/journal.ppat.1003198.s006", "https://dx.doi.org/10.1371/journal.ppat.1003198.s007", "https://dx.doi.org/10.1371/journal.ppat.1003198.s008"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Super_Resolution_Microscopy_Reveals_Specific_Recruitment_of_HIV_1_Envelope_Proteins_to_Viral_Assembly_Sites_Dependent_on_the_Envelope_C_Terminal_Tail__/642500", "title"=>"Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-03-01 14:04:43"}

PMC Usage Stats | Further Information

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Relative Metric

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