Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

University of Texas Medical Branch

university of texas medical branch

&#x5DF2;&#x7ECF;&#x4E3A;&#x76F4;&#x63A5;&#x514B;&#x9686;&#x5916;&#x6E90; dna &#x5BFC;&#x5165;&#x6746;&#x72B6;&#x75C5;&#x6BD2;&#x57FA;&#x56E0;&#x7EC4;&#x514B;&#x9686;&#x7CFB;&#x7EDF;&#x4F53;&#x5916;&#x6746;&#x72B6;&#x75C5;&#x6BD2;&#x3002;&#x8FD9;&#x4E00;&#x7CFB;&#x7EDF;&#x79F0;&#x4E3A;&quot;Homingbac &#x7CFB;&#x7EDF;&quot;&#xFF0C;&#x56E0;&#x4E3A;&#x5B83;&#x4F7F;&#x7528;&#x8FD4;&#x5DE2;&#x5185;&#x5207;&#x9176;&#x3002;Homingbac &#x7CFB;&#x7EDF;&#x88AB;&#x5DE5;&#x7A0B;&#x5230;&#x6746;&#x72B6;&#x75C5;&#x6BD2; AcMNPV&#x3001; &#x5BB6;&#x8695;&#x3001; PxMNPV&#x3001; RoMNPV&#x3001; HaSNPV &#x548C; HzSNPV&#x3002;Homingbac &#x7684;&#x6240;&#x6709;&#x75C5;&#x6BD2;&#x90FD;&#x88AB;&#x90FD;&#x4E3A;&#x4E86;&#x4FDD;&#x7559;&#x7684;&#x591A;&#x9762;&#x4F53;&#x8868;&#x578B;&#xFF0C;&#x4EE5;&#x4FBF;&#x4ED6;&#x4EEC;&#x53EF;&#x4EE5;&#x63A5;&#x79CD;&#x6BCF;&#x5BF9;&#x6606;&#x866B;&#x7684; os&#x3002;&#x8FD9;&#x662F;&#x5E38;&#x89C1;&#x7684;&#x4F53;&#x5916;&#x6606;&#x866B;&#x6746;&#x72B6;&#x75C5;&#x6BD2;&#x514B;&#x9686;&#x7CFB;&#x7EDF;&#x53D6;&#x5F97;&#x4E86;&#x5305;&#x62EC;&#x8FD9;&#x4E24;&#x4E2A;&#x7EC4;&#x7684;&#x591A;&#x4E2A;&#x6606;&#x866B;&#x6746;&#x72B6;&#x75C5;&#x6BD2;&#x7269;&#x79CD;&#x4E00;&#x548C;&#x4E8C; nucleopolyhedroviruses (NPVs) &#x7684;&#x9996;&#x6B21;&#x3002;&#x5728;&#x6B64;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;Homingbac &#x7CFB;&#x7EDF;&#x8868;&#x73B0;&#x76F4;&#x63A5;&#x514B;&#x9686; PCR &#x6269;&#x589E;&#x75C5;&#x7406;&#x6027;&#x57FA;&#x56E0;&#x5361;&#x5E26;&#x5230;&#x7236; Homingbac &#x75C5;&#x6BD2;&#x3002;&#x6B64;&#x65B0;&#x7EC4;&#x96C6;&#x5408;&#x7684;&#x6211;&#x548C;&#x4E8C; NPV Homingbac &#x75C5;&#x6BD2;&#x5927;&#x5927;&#x6269;&#x5C55;&#x4E86;&#x4F53;&#x5916;&#x514B;&#x9686;&#x6280;&#x672F;&#xFF0C;&#x4F7F;&#x91CD;&#x7EC4;&#x6746;&#x72B6;&#x75C5;&#x6BD2;&#x7684;&#x65B0;&#x5DE5;&#x5177;&#x3002;

&#x514B;&#x9686;&#x7CFB;&#x7EDF; Homingbac &#x6746;&#x72B6;&#x75C5;&#x6BD2;&#xFF1A; &#x6606;&#x866B;&#x6746;&#x72B6;&#x75C5;&#x6BD2;&#x57FA;&#x56E0;&#x7EC4;&#x4E2D;&#x5F15;&#x5165;&#x5916;&#x6E90; DNA &#x7684;&#x53E6;&#x4E00;&#x79CD;&#x65B9;&#x6CD5;&#x3002;

An in vitro baculovirus cloning system has been developed for direct cloning of foreign DNA into baculovirus genomes. This system is called the "Homingbac system" because it uses homing endonucleases. The Homingbac system was engineered into the baculoviruses AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV and HzSNPV. All Homingbac viruses were designed to retain the polyhedra phenotype so that they could be inoculated per os to insects. This is the first time a common in vitro baculovirus cloning system has been made for multiple baculovirus species that include both groups I and II nucleopolyhedroviruses (NPVs). In this study, the Homingbac system was demonstrated by directly cloning a PCR-amplified beta-glucuronidase gene cassette into a parent Homingbac virus. This new collection of groups I and II NPV Homingbac viruses are a significant expansion of in vitro cloning technology and are new tools for making recombinant baculoviruses.

The Homingbac baculovirus cloning system: An alternative way to introduce foreign DNA into baculovirus genomes.

Un syst&egrave;me de clonage de baculovirus in vitro a &eacute;t&eacute; d&eacute;velopp&eacute; pour clonage direct d&#39;ADN &eacute;tranger dans les g&eacute;nomes de baculovirus. Ce syst&egrave;me est appel&eacute; le &laquo; syst&egrave;me Homingbac &raquo; car il utilise des endonucl&eacute;ases de radioralliement. Le syst&egrave;me Homingbac a &eacute;t&eacute; con&ccedil;u dans les baculovirus, AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV et HzSNPV. Tous les virus de l&#39;Homingbac ont &eacute;t&eacute; con&ccedil;us pour conserver le ph&eacute;notype de poly&egrave;dres afin qu&#39;ils pourraient &ecirc;tre inocul&eacute;s par os aux insectes. C&#39;est la premi&egrave;re fois, une commune baculovirus in vitro syst&egrave;me de clonage a &eacute;t&eacute; effectu&eacute; pour plusieurs esp&egrave;ces de baculovirus qui incluent les deux groupes I et II nucleopolyhedroviruses (NPN). Dans cette &eacute;tude, le syst&egrave;me Homingbac a &eacute;t&eacute; d&eacute;montr&eacute; par le clonage directement une cassette du g&egrave;ne b&ecirc;ta-glucuronidase amplifi&eacute; par PCR en un virus Homingbac de parent. Cette nouvelle collection des groupes I et II van Homingbac virus sont une expansion significative de la technologie de clonage in vitro et sont de nouveaux outils pour la fabrication des baculovirus recombinants.

Le baculovirus Homingbac syst&egrave;me de clonage : une autre fa&ccedil;on d&#39;introduire de l&#39;ADN &eacute;tranger dans les g&eacute;nomes de baculovirus.

Eine in-vitro Baculovirus Klonen System wurde entwickelt f&uuml;r direkte Klonen fremder DNA in Baculovirus-Genome. Dieses System ist das &quot;Homingbac System&quot; genannt, weil zielsuchende Endonucleases verwendet wird. Das Homingbac-System wurde in den Baculoviruses AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV und HzSNPV entwickelt. Alle Homingbac-Viren wurden entwickelt, um das Polyeder-Ph&auml;notyp zu behalten, so dass sie pro os zu Insekten geimpft werden konnte. Dies ist das erste Mal eine gemeinsame in-vitro Baculovirus Klonen System erzielt wurden f&uuml;r mehrere Baculovirus-Arten, die beide Gruppen umfassen I und II Nucleopolyhedroviruses (NPVs). In dieser Studie zeigte sich das Homingbac-System von Klonen direkt eine PCR verst&auml;rkt Beta-Glukuronidase-gen-Kassette in einen &uuml;bergeordneten Homingbac Virus. Diese neue Kollektion der Gruppen I und II NBW Homingbac Viren sind eine deutliche Ausweitung der in-vitro-Klonen-Technologie und neue Tools f&uuml;r die Herstellung von rekombinanten Baculoviruses.

Die Homingbac-Baculovirus Klonen System: eine alternative M&ouml;glichkeit, fremden DNA in Baculovirus Genome einzuf&uuml;hren.

Un baculovirus in vitro, clonazione sistema &egrave; stato sviluppato per clonazione diretta di DNA estraneo in baculovirus genomi. Questo sistema &egrave; chiamato il &quot;sistema Homingbac&quot; perch&eacute; utilizza homing endonucleases. Il sistema Homingbac &egrave; stato progettato in baculoviruses AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV e HzSNPV. Tutti i virus di Homingbac sono stati progettati per mantenere il fenotipo di poliedri che essi potevano essere inoculati per os per insetti. Questa &egrave; la prima volta un comune baculovirus in vitro, clonazione sistema &egrave; stata fatta per molteplici specie di baculovirus che includono entrambi i gruppi I e II nucleopolyhedroviruses (NPVs). In questo studio, il sistema Homingbac &egrave; stato dimostrato da clonazione direttamente una cassetta del gene beta-glucuronidasi PCR amplificati in un virus Homingbac padre. Questa nuova collezione di gruppi I e II NPV Homingbac virus sono una significativa espansione della tecnologia di clonazione in vitro e nuovi strumenti per fare baculoviruses ricombinante.

Il baculovirus Homingbac sistema di clonazione: un modo alternativo per introdurre DNA estraneo in baculovirus genomi.

&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x4F5C;&#x6210;&#x3001;in vitro &#x3067;&#x306E;&#x30D0;&#x30AD;&#x30E5;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30D0;&#x30AD;&#x30E5;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306B;&#x5916;&#x6765; DNA &#x306E;&#x76F4;&#x63A5;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306E;&#x305F;&#x3081;&#x306B;&#x958B;&#x767A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30DB;&#x30FC;&#x30DF;&#x30F3;&#x30B0;&#x30A8;&#x30F3;&#x30C9;&#x30CC;&#x30AF;&#x30EC;&#x30A2;&#x30FC;&#x30BC;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x306E;&#x3067;&#x3053;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x300C;Homingbac &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x300D;&#x3068;&#x547C;&#x3070;&#x308C;&#x307E;&#x3059;&#x3002;Homingbac &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306F;&#x3001;AcMNPV, &#x4E00;&#x822C;&#x8B1B;&#x6F14;, PxMNPV&#x3001;RoMNPV&#x3001;HaSNPV&#x3001;HzSNPV &#x306E;&#x30D0;&#x30AD;&#x30E5;&#x30ED;&#x306B;&#x8A2D;&#x8A08;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3059;&#x3079;&#x3066;&#x306E; Homingbac &#x30A6;&#x30A4;&#x30EB;&#x30B9; os &#x6606;&#x866B;&#x306B;&#x3042;&#x305F;&#x308A;&#x306B;&#x63A5;&#x7A2E;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x3088;&#x3046;&#x306B;&#x3001;&#x591A;&#x9762;&#x4F53;&#x306E;&#x8868;&#x73FE;&#x578B;&#x3092;&#x4FDD;&#x6301;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x8A2D;&#x8A08;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x306F;&#x3001;&#x3057;&#x3001;II &#x8CAA;&#x98DF; (Npv) &#x4E21;&#x65B9;&#x306E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x3092;&#x542B;&#x3081;&#x308B;&#x305F;&#x3081;&#x306E;&#x30D0;&#x30AD;&#x30E5;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x7A2E;&#x306F;&#x8907;&#x6570;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x4F5C;&#x6210;&#x4E00;&#x822C;&#x7684;&#x306A; in vitro &#x30D0;&#x30AD;&#x30E5;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x304C;&#x884C;&#x308F;&#x308C;&#x305F;&#x306E;&#x521D;&#x3081;&#x3066;&#x3067;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;Homingbac &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3001;&beta;-&#x30B0;&#x30EB;&#x30AF;&#x30ED;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x306E; PCR &#x5897;&#x5E45;&#x907A;&#x4F1D;&#x5B50;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#x3092;&#x89AA; Homingbac &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306B;&#x76F4;&#x63A5;&#x8907;&#x88FD;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x793A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x3057;&#x3001;II NPV Homingbac &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306F; in vitro &#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x6280;&#x8853;&#x306E;&#x5927;&#x5E45;&#x306A;&#x62E1;&#x5927;&#x3068;&#x7D44;&#x63DB;&#x3048;&#x30D0;&#x30AD;&#x30E5;&#x30ED;&#x3092;&#x4F5C;&#x308B;&#x305F;&#x3081;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30C4;&#x30FC;&#x30EB;&#x3067;&#x3059;&#x3002;

&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x4F5C;&#x6210; Homingbac &#x30D0;&#x30AD;&#x30E5;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;: &#x30D0;&#x30AD;&#x30E5;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306B;&#x5916;&#x6765; DNA &#x3092;&#x5C0E;&#x5165;&#x3059;&#x308B;&#x5225;&#x306E;&#x65B9;&#x6CD5;&#x3002;

&#xC2DC;&#xC2A4;&#xD15C; &#xBCF5;&#xC81C; &#xCCB4; &#xC678; &#xC7A0;&#xC7AC; &#xC9C1;&#xC811; &#xC7A0;&#xC7AC; &#xC720;&#xC804;&#xC790;&#xC5D0; &#xC678;&#xB798; DNA&#xC758; &#xD074;&#xB85C;&#xB2DD;&#xC744; &#xC704;&#xD574; &#xAC1C;&#xBC1C; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC2DC;&#xC2A4;&#xD15C; &#xCD94;&#xC801; endonucleases&#xC744; &#xC0AC;&#xC6A9; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &quot;Homingbac &#xC2DC;&#xC2A4;&#xD15C;&quot; &#xC774;&#xB77C;&#xACE0; &#xD569;&#xB2C8;&#xB2E4;. Homingbac &#xC2DC;&#xC2A4;&#xD15C; baculoviruses AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV &#xBC0F; Hzsnpv&#xB85C; &#xC124;&#xACC4; &#xB410;&#xB2E4;. &#xBAA8;&#xB4E0; Homingbac &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xB294; &#xACE4;&#xCDA9;&#xC5D0; os &#xB2F9; &#xC8FC;&#xC0AC; &#xB420; &#xC218; &#xC788;&#xB3C4;&#xB85D; &#xB2E4;&#xBA74;&#xCCB4; &#xD615;&#xC744; &#xC720;&#xC9C0; &#xD558;&#xB3C4;&#xB85D; &#xC124;&#xACC4; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xAC83;&#xC740; &#xCC98;&#xC74C;&#xC73C;&#xB85C; &#xACF5;&#xD1B5; &#xCCB4; &#xC678; &#xC7A0;&#xC7AC; &#xC2DC;&#xC2A4;&#xD15C; &#xBCF5;&#xC81C; &#xD588;&#xB2E4; &#xB450; &#xADF8;&#xB8F9;&#xC744; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xC5EC;&#xB7EC; &#xC7A0;&#xC7AC; &#xC885;&#xC871;&#xC5D0; &#xB300; &#xD55C; I&#xC640; II nucleopolyhedroviruses (NPVs). &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C;&#xB294; Homingbac &#xC2DC;&#xC2A4;&#xD15C;&#xC740; &#xC9C1;&#xC811; &#xBD80;&#xBAA8; Homingbac &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC5D0; PCR &#xC99D;&#xD3ED; &#xBCA0;&#xD0C0;-glucuronidase &#xC720;&#xC804;&#xC790; &#xCE74;&#xC138;&#xD2B8;&#xB97C; &#xBCF5;&#xC81C; &#xD558; &#xC5EC; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. &#xC774; &#xC0C8;&#xB85C;&#xC6B4; &#xADF8;&#xB8F9; &#xCEEC;&#xB809;&#xC158; I &#xBC0F; II NPV Homingbac &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xCCB4; &#xC678; &#xBCF5;&#xC81C; &#xAE30;&#xC220;&#xC758; &#xC0C1;&#xB2F9;&#xD55C; &#xD655;&#xC7A5; &#xC774;&#xBA70; &#xC7AC;&#xC870;&#xD569; baculoviruses &#xB9CC;&#xB4E4;&#xAE30; &#xC704;&#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xB3C4;&#xAD6C; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xBCF5;&#xC81C; &#xC2DC;&#xC2A4;&#xD15C; Homingbac &#xC7A0;&#xC7AC;: &#xC7A0;&#xC7AC; &#xC720;&#xC804;&#xC790;&#xB85C; &#xC678;&#xAD6D; DNA &#xC18C;&#xAC1C; &#xD558;&#xB294; &#xB2E4;&#xB978; &#xBC29;&#xBC95;.

Un baculovirus in vitro clonaci&oacute;n sistema ha sido desarrollado para clonaci&oacute;n directa de ADN extra&ntilde;o en los genomas de baculovirus. Este sistema se llama el &quot;sistema Homingbac&quot; ya que utiliza homing Endonucleasas. El sistema de Homingbac fue dise&ntilde;ado en el baculovirus, AcMNPV, BmNPV, PxMNPV, RoMNPV, HaSNPV y HzSNPV. Todos los virus de Homingbac fueron dise&ntilde;ados para retener el fenotipo de poliedros, por lo que podr&iacute;a ser inoculadas por os a los insectos. Esta es la primera vez que un baculovirus in vitro com&uacute;n sistema de clonaci&oacute;n se ha hecho para varias especies de baculovirus que incluyen ambos grupos I y II nucleopolyhedroviruses (NPVs). En este estudio, el sistema Homingbac fue demostrado por clonaci&oacute;n directamente un cassette de polimerizaci&oacute;n en cadena-amplificado beta-glucuronidase gen en un virus de la Homingbac de los padres. Esta nueva colecci&oacute;n de los grupos I y II NPV Homingbac virus son una significativa expansi&oacute;n de la tecnolog&iacute;a de clonaci&oacute;n in vitro y son nuevas herramientas para la fabricaci&oacute;n de baculovirus recombinante.

Los baculovirus Homingbac sistema de clonaci&oacute;n: una manera alternativa para introducir ADN extra&ntilde;o en los genomas de baculovirus.

&#x88C2;&#x8C37;&#x70ED;&#x75C5;&#x6BD2; (RVFV &#xFF1B; &#x5BB6;&#x5EAD; Bunyaviridae&#xFF0C;&#x5C5E; Phlebovirus) &#x662F;&#x91CD;&#x8981;&#x7684;&#x4EBA;&#x7C7B;&#x548C;&#x53CD;&#x520D;&#x52A8;&#x7269;&#x7684;&#x65B0;&#x5174;&#x75C5;&#x539F;&#x4F53;&#x3002;&#x5176; NSs &#x86CB;&#x767D;&#x4EE5;&#x524D;&#x5DF2;&#x88AB;&#x5217;&#x4E3A;&#x4E3B;&#x8981;&#x7684;&#x6BD2;&#x529B;&#x56E0;&#x5B50;&#xFF0C;&#x901A;&#x8FC7;&#x4E09;&#x4E2A;&#x4E0D;&#x540C;&#x7684;&#x673A;&#x5236;&#x6291;&#x5236;&#x5BBF;&#x4E3B;&#x9632;&#x5FA1;&#xFF1A; &#x5B83;&#x76F4;&#x63A5;&#x6291;&#x5236; &beta; &#x5E72;&#x6270;&#x7D20; (&#x5E72;&#x6270;&#x7D20; &beta;) &#x542F;&#x52A8;&#x5B50;&#x6D3B;&#x6027;&#xFF0C;&#x5B83;&#x4FC3;&#x8FDB;&#x9000;&#x5316;&#x7684;&#x53CC;&#x94FE; RNA &#x4F9D;&#x8D56;&#x7684;&#x86CB;&#x767D;&#x6FC0;&#x9176; (&#x6551;&#x6D4E;&#x6B3E;) &#x548C;&#x6270;&#x4E71;&#x5927;&#x4F1A;&#x901A;&#x8FC7;&#x87AF;&#x5408;&#x4F5C;&#x7528;&#x5176; p44 &#x4E9A;&#x5355;&#x4F4D;&#x7684;&#x57FA;&#x5E95;&#x8F6C;&#x5F55;&#x56E0;&#x5B50; TFIIH&#xFF0C;&#x5B83;&#x538B;&#x5236;&#x4E3B;&#x673A;&#x8F6C;&#x5F55;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x9664;&#x4E86;&#x6551;&#x6D4E;&#x6B3E;&#xFF0C;NSs &#x4E5F;&#x4FC3;&#x8FDB; TFIIH &#x4E9A;&#x57FA; p62 &#x7684;&#x9000;&#x5316;&#x3002;RVFV MP 12 &#x75AB;&#x82D7;&#x682A;&#x7EC6;&#x80DE;&#x611F;&#x67D3;&#x51CF;&#x5C11; p62 &#x86CB;&#x767D;&#x6C34;&#x5E73;&#x5BF9;&#x521D;&#x671F;&#x611F;&#x67D3;&#x68C0;&#x6D4B;&#x9650;&#x5EA6;&#x4EE5;&#x4E0B;&#x3002;&#x6B64; NSs &#x4ECB;&#x5BFC; p62 &#x7684;&#x4E0B;&#x8C03;&#x662F;&#x7FFB;&#x8BD1;&#x540E;&#xFF0C;&#x5B83;&#x4E0D;&#x53D7;&#x6291;&#x5236;&#x8F6C;&#x5F55;&#x6216;&#x7FFB;&#x8BD1;&#x7684;&#x836F;&#x7406;&#x4F5C;&#x7528;&#x548C; MP 12 &#x611F;&#x67D3;&#x4E86; p62 mRNA &#x6C34;&#x5E73;&#x6CA1;&#x6709;&#x5F71;&#x54CD;&#x3002;&#x7EC6;&#x80DE;&#x4E0E;&#x86CB;&#x767D;&#x9176;&#x4F53;&#x6291;&#x5236;&#x5242;&#x7684;&#x6CBB;&#x7597;&#xFF0C;&#x4F46;&#x4E0D;&#x662F;&#x6291;&#x5236;&#x6EB6;&#x9176;&#x4F53;&#x9178;&#x5316;&#x6216;&#x6838;&#x51FA;&#x53E3;&#x5BFC;&#x81F4;&#x4E0B; NSs p62 &#x8D8B;&#x4E8E;&#x7A33;&#x5B9A;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x53EF;&#x4EE5;&#x4ECE;&#x53D7;&#x611F;&#x67D3;&#x7EC6;&#x80DE;&#x88C2;&#x89E3;&#x4E0E; NSs coprecipitated p62&#x3002;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E; RVFV NSs &#x86CB;&#x767D;&#x662F;&#x80FD;&#x591F;&#x4E0E;&#x53D7;&#x611F;&#x67D3;&#x7EC6;&#x80DE;&#x5185; TFIIH &#x4E9A;&#x57FA; p62 &#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#xFF0C;&#x5E76;&#x4FC3;&#x8FDB;&#x5176;&#x9000;&#x5316;&#xFF0C;&#x90FD;&#x53EF;&#x4EE5;&#x76F4;&#x63A5;&#x5728;&#x7EC6;&#x80DE;&#x6838;&#x4E2D;&#x51FA;&#x73B0;&#x3002;

NSs &#x7684;&#x88C2;&#x8C37;&#x70ED;&#x75C5;&#x6BD2;&#x86CB;&#x767D;&#x4FC3;&#x8FDB;&#x7FFB;&#x8BD1;&#x540E;&#x4E0B;&#x8C03;&#x7684; TFIIH &#x4E9A;&#x57FA; p62&#x3002;

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-Î²) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

Rift Valley fever virus (RVFV), belonging to the genus Phlebovirus, family Bunyaviridae, is endemic to sub-Saharan Africa and causes a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. MP-12 is the only RVFV strain excluded from the select-agent rule and handled at a biosafety level 2 (BSL2) laboratory. MP-12 encodes a functional major virulence factor, the NSs protein, which contributes to its residual virulence in pregnant ewes. We found that 100% of mice subcutaneously vaccinated with recombinant MP-12 (rMP12)-murine PKRN167 (mPKRN167), which encodes a dominant-negative form of mouse double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in place of NSs, were protected from wild-type (wt) RVFV challenge, while 72% of mice vaccinated with MP-12 were protected after challenge. rMP12-mPKRN167 induced alpha interferon (IFN-Î±) in sera, accumulated RVFV antigens in dendritic cells at the local draining lymph nodes, and developed high levels of neutralizing antibodies, while parental MP-12 induced neither IFN-Î± nor viral-antigen accumulation at the draining lymph node yet induced a high level of neutralizing antibodies. The present study suggests that the expression of a dominant-negative PKR increases the immunogenicity and efficacy of live-attenuated RVFV vaccine, which will lead to rational design of safe and highly immunogenic RVFV vaccines for livestock and humans.

The dominant-negative inhibition of double-stranded RNA-dependent protein kinase PKR increases the efficacy of Rift Valley fever virus MP-12 vaccine.

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-Î² mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2Î± through PKR activation, while retaining the function to inhibit host general transcription including IFN-Î² gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2Î±, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2Î± in infected cells undergoing host general transcription suppression.

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

Modifying the NSs gene to improve live-attenuated vaccine for Rift Valley fever.

Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-Î² gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.

Rift Valley fever virus MP-12 vaccine encoding Toscana virus NSs retains neuroinvasiveness in mice.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-Î² promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-Î² mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever.

Olga Lihoradova

Department of Pathology,
Sealy Center for Vaccine Development,
Center for Biodefense and Emerging Infectious Diseases,
Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases

University of Texas Medical Branch

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis