אנו מודים יוהיי Yamauchi ורוברטה מנצ&#39;יני לספק לנו חומרים כימיים. מעבדת AH נתמכה על ידי רשתות ההדרכה הראשוניות מארי קירי (ITN), מועצת המחקר האירופית (ERC), ועל ידי הקרן הלאומית למדע השויצרי (Sinergia).

1. הכנת חוצצים ופתרונות במלאי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן חיץ MNT (20 MES מ&quot;מ, 30 מ&quot;מ טריס ו 100 mM NaCl) על ידי המסת 470 מ&quot;ג טריס Hydrochloride, 390 מ&quot;ג מימה MES ו 580 מ&quot;ג NaCl ב 80 מ&quot;ל DDH 2 O. התאם את חיץ pH 7.4 ולהביא אותו לנפח סופי של 100 מ&quot;ל עם DDH 2 O. </li><li style=";text-align:right;direction:rtl"> הכן שלושה פתרונות זהים של 500 חיץ מ&quot;מ MES על ידי המסת מימה MES 9.76 גרם 80 מ&quot;ל DH 2 O כל אחד. התאם את מאגרי 5.8 pH, 5.4, ו -5.0, בהתאמה, על ידי titrating עם NaOH המרוכז. תביאו כל חיץ לנפח סופי של 100 מ&quot;ל עם DH 2 O. </li><li style=";text-align:right;direction:rtl"> הכן שני פתרונות זהים של 500 מ&quot;מ טריס חיץ ידי המסת 7.88 גרם טריס הידרוכלוריד ב 80 מ&quot;ל DH 2 O כל ולהתאים את ה- pH ל 7.4 ו -6.4, בהתאמה, על ידי titrating עם מרוכזת HCI. תביאו למאגר לנפח סופי של 100 מ&quot;ל עם DH 2 O. </li><li style=";text-align:right;direction:rtl"> הכן פתרון גליצרול 50% ב DH 2 O ומערבבים היטב עד glycארול הוא מעורב homogenously. סנן את הפתרון באמצעות יחידת מסנן Steritop (0.22 מיקרומטר גודל נקבובי) לתוך בקבוק זכוכית. </li><li style=";text-align:right;direction:rtl"> כן 10% פתרון NP-40 ו 1 M NaCl ב DH 2 O. </li><li style=";text-align:right;direction:rtl"> הכן 25x מעכב פרוטאז מרוכז על ידי המסת שתי טבליות ב 4 מ&quot;ל DDH 2 O. </li></ol> 2. הכנת מעברי צבע גליצרול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כן 5 מיליליטר לערבב חומרי ניקוי החיץ מאסטר (300 mM NaCl, 2% NP-40, 2x מרוכז מעכבי פרוטאז) פתרון עבור כל תנאי pH לבדוק (pH 7.4, 6.4, 5.8, 5.4, ו -5.0). לשם כך, לערבב 9 מ&quot;ל של 1 M NaCl, 6 מ&quot;ל של 10% NP-40, 2.4 מ&quot;ל של המניה מעכב פרוטאז 25x, ו- 9 מ&quot;ל DDH 2 0. </li><li style=";text-align:right;direction:rtl"> עבור כל pH מצב pipet 4.4 מ&quot;ל של מיקס מאסטר לתוך צינור חרוטי 50 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> עבור ערכי ה- pH מעל 5.8 להוסיף 0.6 מ&quot;ל של פתרון המניות בהתאמה מותאם pH 500 מ&quot;מ טריס אל הצינור. עבור 5.8 pH והתחתון להוסיף 0.6 מ&quot;ל של פתרון המניות pH המותאמת בהתאמה 500 מ&quot;מ MES. </li><li style=";text-align:right;direction:rtl"> הפוך 5מ&quot;ל של תמיסת מאגר המכיל 300 mM NaCl, מעכבי פרוטאז 2x, 60 מ&quot;מ טריס מותאם pH 7.4 ו DDH 2 O. זה ישמש חיץ שיפוע מלא חומר ניקוי חינם. </li><li style=";text-align:right;direction:rtl"> במידת הצורך, עדין לכוונן את ה- pH של פתרונות pH 7.4, 6.4, 5.8, 5.4, ו -5.0 ידי הוספת פתרונות HCI או NaOH מרוכז, בהתאמה. </li><li style=";text-align:right;direction:rtl"> הוסף 5 מ&quot;ל של המניה גליצרול 50% עד 5 מ&quot;ל של המכילים חומרי ניקוי וחומרי ניקוי ללא תערובות חיץ וכתוצאה מכך שישה פתרונות גליצרול שונים 25%. בדוק את ערכי ה- pH באמצעות רצועות אינדיקטור pH. הערה: במידה pH נמדד שונה באופן משמעותי מן הערך הרצוי, הפתרונות המתאימים צריכים להיות מוכנים שוב. </li><li style=";text-align:right;direction:rtl"> כן פתרון גליצרול 15% על ידי ערבוב 50% מניות גליצרול עם DH 2 O ב 3: יחס 7. </li></ol> 3. ultracentrifugation של IAV <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 3 פתרון גליצרול מ&quot;ל 15% לתוך צינורות צנטריפוגה אולטרה ברור (13.2 מ&quot;ל, 14 מ&quot;מ x 89 מ&quot;מ) באמצעות מזרק 5 מ&quot;ל ו לביתDLE (21 G, 9 ס&quot;מ). אין להשאיר טיפות על הקיר הפנימי של הצינור, שכן הדבר עלול להפריע את היושרה של השיפוע. חזור על פעולה זו עבור סכום כולל של שישה צינורות צנטריפוגה, אחד עבור כל אחד מחמשת התנאים pH לבדוק ואחד למדגם שליטה. </li><li style=";text-align:right;direction:rtl"> בזהירות במקום 3.4 מ&quot;ל 25% פתרון גליצרול מתחת לשכבת גליצרול 15% באמצעות מזרק 5 מ&quot;ל ו מחט ארוכה. יש להיזהר שלא לערבב את שתי השכבות. חזור על פעולה זו עבור כל ששת התנאים לבדוק עם פתרונות גליצרול pH המותאמת בהתאמה. הערה: עבודת בארון biosafety בכיתה השנייה על הצעדים הבאים. </li><li style=";text-align:right;direction:rtl"> בעדינות כיסוי הדרגתי גליצרול עם 30 μl בהירים נוזל allantoic המכיל IAV (X31, H3N2) מדולל 1 מיליליטר חיץ MNT (מקביל סביב 20-30 מיקרוגרם של חלבון נגיפי הכולל) עבור כל צבע. </li><li style=";text-align:right;direction:rtl"> יתרה מנוגדים צינורות ומניחים אותם לתוך הרוטור ultracentrifugation SW41. צנטריפוגה עבור 150 דקות, ב 55,000 XG, ו -12 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לאחר צנטריפוגה, בזהירותלהסיר את supernatant, כלומר, הן שכבות גליצרול באמצעות פיפטה פסטר נקייה וכן aspirator. Resuspend גלולה ב 40 μl (1x) חיץ מדגם LDS הלא צמצום. חשוב pipet מעלה ומטה מספר פעמים כדי לפזר את הגלולה לחלוטין. מעבירים את המדגם לתוך צינור 1.5 מ&quot;ל microcentrifuge. </li></ol> 4. SDS-PAGE של גלולה שברים ו Coomassie מכתים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מחמם את כל הדגימות ב 95 מעלות צלזיוס במשך 10 דקות. בשלב זה הדגימות ניתן לאחסן ב -20 מעלות צלזיוס עד שהם מנותחים על ידי SDS-PAGE. </li><li style=";text-align:right;direction:rtl"> טען 20 μl של כדורי מומס על שיפוע מראש יצוק (4-12%) Bis-טריס ג&#39;ל מיני ולהפעיל עבור שעה 1 ב 200 V ב 1x מגבים SDS הפעלת המאגר. </li><li style=";text-align:right;direction:rtl"> בצע פתרון קיבעון עם 40% מתנול 10% קרחוני חומצה אצטית DDH 2 O. </li><li style=";text-align:right;direction:rtl"> דגירה ג&#39;ל בתמיסה קיבעון עבור שעה 1 ו להכתים לילה בצלחת תרבית תאים 15 ס&quot;מ עם נפח מספיק של פתרון Coomassie קולואידים בעוד בעדינותרועד בטמפרטורת החדר. הערה: חשוב לסגור את המנה על מנת למנוע אידוי של הפתרון המכתים. </li><li style=";text-align:right;direction:rtl"> Destain הג&#39;ל DDH 2 O. החזר את DDH 2 O כל 15-20 דקות עד שרקע ג&#39;ל מתבהר. אחסן את הג&#39;ל DDH 2 O על 4 מעלות צלזיוס עד שהוא נסרק כימות הלהקה. </li><li style=";text-align:right;direction:rtl"> סרוק את הג&#39;ל על ברזולוציה גבוהה ולהשתמש תוכנה זמינה או מחוייט מסחרית עבור כימות של עוצמות להקת חלבון. הפחת את אות רקע מאזור קרוב להקות בהתאמה לנרמל את הערכים הללו ל דגימות בקרת חומר הניקוי ללא (ב- pH 7.4). </li></ol>

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The authors have nothing to disclose.

capsids ויראלי הם קומפלקסים macromolecular metastable. בעוד הרכבה של virions מחייבת encapsidation ועיבוי של הגנום הווירוס, ייזום של הסיבוב הבא של זיהום תלוי פירוק מבנה קפסיד קומפקטי זו. וירוסים התפתחו לנצל מנגנונים תאיים שונים כדי לשלוט על מחזור הציפוי-uncoating, כולל קולטני תאים, מלווה, אנזימים פ...

וירוס שפעת A (IAV) הוא וירוס אפוף ושייך למשפחת Orthomyxoviridae. הגנים שלו מקודדים על גנום RNA מפולח, שלילי-השכל חד-גדילים. אצל בני אדם, IAV גורם זיהומים בדרכי נשימה, אשר מתרחשים התפרצויות מגיפה עונתיות ונושאת פוטנציאל המגיפות העולמיות 1. עם קשירת שאריות חומצת sialic על תא מארח המשטח 2, IAV מופנם על ידי אנדוציטוזה clathrin התלוי מסלולים עצמאיים-clathrin 3-8. המילייה חומצי (pH &lt;5.5) ב vacuoles endocytic מפעילה שינוי קונפורמציה מרכזי hemagglutinin גליקופרוטאין ספייק IAV (HA), שתוצאתה היתוך של נגיפי ואת endosomal מאוחר קרום 9. לאחר קפסיד IAV (כאן המכונה גם ויראלי &quot;הליבה&quot;) ברח מן endosomes מאוחר (LES), הוא ללא ציפוי ב cytosol ואחריו התחבורה של ribonucleoproteins ויראלי (vRNPs) לתוך הגרעין - O את האתרf וירוס שכפול שעתוק 10-13. לפני הפעלת חומצה של HA בנגיף חווה ירידה הדרגתית pH במערכת endocytic, אשר מספרים ראשוניים הליבה עבור פירוקו לאחר 14-16. בחודש זה &quot;הטרמה&quot; צעד של תעלות יונים M2 בקרום ויראלי לתווך זרם של פרוטונים ו- K +10,14,16. השינוי בריכוז יוני בפנים הווירוס מפריע האינטראקציות להצטבר על ידי חלבון מטריקס ויראלי M1 ואת צרור השמונה vRNP, ומקל IAV uncoating בציטופלסמה הבא קרום היתוך 10,14-17. ניתוח ישיר וכמותיים של הצעד יחול כבר הקשה על ידי העובדה endosomes קשה לגשת באופן ניסיוני. תהליך הכניסה, יתר על כן, מאוד לא סינכרוני. בנוסף, מבחני סוף נקודות, כגון qRT-PCR של מדידות infectivity או רנ&quot;א נגיפי שוחרר, אינם מספקים תמונה מפורטת על מצב ביוכימי של capsi ויראליד בכל צעד נתון כניסה. בעוד ההפרעות של endosomes ידי siRNA או טיפול תרופתי תרם משמעותית להבנת כניסה IAV 18-20, כיוונון עדין קשה ונוטים תופעות לוואי נוקבים בתוכנית התבגרות אנדוזום תחת פיקוח הדוק. כדי למנוע בעיות אלה, יש לנו התאמנו שפותח בעבר בפרוטוקול במבחנה מבוסס על שימוש צנטריפוגה שיפוע מהיר 17. בניגוד לניסיונות אחרים 21,22, 23,24, שהתבססו בעיקר על שילובים של מחשוף פרוטאוליטים וטיפול חומר ניקוי ואחריו ניתוח EM, גישה זו אפשרה להם תוצאה לכימות בקלות. צנטריפוגה דרך שכבות צבע שונות מאפשרת החלקיקים לסדימנטציה להיחשף ומגיבים עם שינוי תנאים באופן מבוקר. בפרוטוקול שהוצג, IAV נגזר נוזל allantoic בהיר או חלקיקים נגיפיים מטוהרים שקועים גליצרול דו שכבתישיפוע שבו השכבה התחתונה מכילה את הלא יוניים חומר ניקוי NP-40 (איור 1). כמו הנגיף חודר את השכבה השנייה, המכיל חומר ניקוי, המעטפה שומנים ויראלי גליקופרוטאינים המעטפה הם solubilized בעדינות והשאיר אחריו. הליבה, מורכב משמונה צרור vRNP ומוקף שכבת מטריקס, משקע כמבנה יציב לתוך השבר הגלול. חלבונים הליבה ויראלי, כגון M1 ו- nucleoprotein הקשורים vRNP (NP), יכול להיות מזוהים את הכדור על ידי SDS-PAGE ו Coomassie מכתים. בפרט, תוך ניצול של ג&#39;ל שיפוע הזמין המסחרי מכתימה עם קולואידים רגישות הגבוהה Coomassie 25, מאפשר דיוק וזיהוי גבוהים של כמויות קטנות אפילו של חלבוני ליבה קשורה ויראלי. זו מגדירה את הבסיס לבדוק האם תנאים שונים כגון pH, ריכוז מלח, וגורם uncoating המשוערת יש השפעות על התנהגות השקיעה של רכיבי ליבת הליבה stabil<html> ity. למטרה זו, רק את השכבה התחתונה, המכיל חומר ניקוי שיפוע גליצרול היא שונה על ידי החדרת הגורם או מצב עניינים. הטכניקה כבר יקרה במיוחד בחקירת ההשפעה שונה ערכי pH וריכוז מלח על שלמות ליבת IAV 16. פעולות ביניים של uncoating IAV יכול להיות במעקב דיסוציאציה כולל של שכבת מטריקס ב- pH חומצי קל (&lt;6.5) ואחריו דיסוציאציה vRNP ב- pH 5.5 והתחתון 16,17 (איור 1). השלב האחרון היה עוד יותר משופר על ידי נוכחות של גבוה K + ריכוז בשכבת גליצרול, המשקף בסביבה endocytic מאוחר 16. לכן, כמו הווירוס ואת הליבה משקעים דרך השיפוע שחווה בסביבה משתנית שתנאים מחקו ב endosomes. התוצאה היתה פירוק הדרגתי של הליבה ויראלי במבחנה, המשלימה התוצאות הנגזרות מבחני ביולוגיה של התא. t &quot;&gt; השיטה המוצגת כאן אפשרה מהר וניתוח לשחזור ביותר של IAV (X31 ו- A / WSN / 33) ואת IBV (B / Lee / 40) uncoating מופעלות על ידי pH חומצי והגדלת K +16,17 וכן פירוק ליבות paramyxovirus בחשיפה pH בסיסי 17. זה מתקבל על הדעת כי ניתן להתאים את הגישה וירוסים אפוף אחרים כדי להשיג תובנות את התכונות הביוכימיות של המבנה קפסיד ויראלי ופירוק קפסיד במהלך ערך בתא.

<table><tbody><tr><td>cOmplete&amp;trade;, EDTA-free protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>11873580001</td><td>The stock solution can be stored at 2 to 8 &amp;deg;C for 1 to 2 weeks</td></tr><tr><td>Glacial acetic acid</td><td>Merck Millipore</td><td>100063</td><td></td></tr><tr><td>Glycerol anhydrous BioChemica</td><td>AppliChem</td><td>A1123</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck Millipore</td><td>100317</td><td></td></tr><tr><td>Long injection needle (21&amp;nbsp;G, 9 cm, bevel or blunt-end)</td><td></td><td></td><td></td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>I8896</td><td>Now commercially available as IGEPAL&amp;reg; CA-630</td></tr><tr><td>NuPAGE 4-12% Bis-Tris mini gels, 10&amp;nbsp;wells, 1.0 mm</td><td>Life Technologies</td><td>NP0321</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Technologies</td><td>NP0008</td><td></td></tr><tr><td>NuPAGE MOPS SDS running buffer (20x)</td><td>Life Technologies</td><td>NP0001</td><td></td></tr><tr><td>pH indicator strips, pH 4.0-7.0</td><td>Merck Millipore</td><td>109542</td><td></td></tr><tr><td>QC Colloidal Coomassie Stain</td><td>BIO RAD</td><td>1610803</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>106406</td><td></td></tr><tr><td>Sodiumhydroxide</td><td>Merck Millipore</td><td>106498</td><td></td></tr><tr><td>Steritop filter unit</td><td>Merck Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>SW41 Ti, ultracentrifuge rotor set</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>Thinwall, Ultra-clear centrifuge tubes, 13.2&amp;nbsp;ml, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Tris hydrochloride&amp;nbsp;</td><td>AppliChem</td><td>A1087</td><td></td></tr><tr><td>X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td><td>Virapur</td><td></td><td>Freshly thawed on 4 &amp;deg;C</td></tr></tbody></table>

in vitro disassembly of influenza a virus capsids by gradient centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

כפי שכבר דן, יחול ב endosomes נדרש כדי להבהיר את ליבת IAV uncoating מוסמכת. Protonation של הליבה מחליש אינטראקציה של M1 ו- vRNPs (מורכב של רנ&quot;א הנגיפי, NP, ואת פולימראז המורכב PB1 / PB2 / רש&quot;פ). תהליך זה מופעל כאשר וירוס נכנס חשוף pH של 6.5 (או נמוכים) ב endosomes מוקדם (EES) וממשיך עד נ...

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

במבחנה פירוק של וירוס שפעת capsids ידי צנטריפוגה שיפוע

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K Views.  ETH Zurich. Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Video: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

ההכנה של ה-DNA ויראלי של Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

ב Uncoating במבחנה של HIV-1 ליבות

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

פשוט וחזק In vivo ו במבחנה גישה ללימוד הרכבה וירוס

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

זיקה טיהור של נגיף השפעת תסביכים Ribonucleoprotein של הכרומטין של תאים נגועים

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

טיהור ויזואליזציה של שפעת מכלולים Ribonucleoprotein ויראלי

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Biology

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

שיטות אלטרנטיביות במבחנה מצפני Capsid ויראלי המבנית

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

הרכבה ולטיהור Intasomes וירוס קצפית אב-טיפוס

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

הליך יעיל וסטנדרטי ליצירת וקטורי וירוסים הקשורים באדנו באיכות גבוהה ובאיכות גבוהה תוך שימוש בפלטפורמת מפעל תאים

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

Bioengineering

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

תא בודד מיקרו-שאיפה כאסטרטגיה חלופית מיון תאים פלואורסצנטית מופעל עבור הפרדה וירוס ענק תערובת

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

בידוד AAV על ידי צנטריפוגה הדרגתית צפיפות יודיקסנול כפולה

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Author Spotlight: בידוד יעיל של וירוסים הקשורים באדנו ליישומים פרה-קליניים

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.