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

&#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2; (HCMV) &#x75C5;&#x6BD2;&#x4F53;&#x5927;&#x4F1A;&#x5728;&#x7EC6;&#x80DE;&#x6838;&#x548C;&#x7EC6;&#x80DE;&#x8D28;&#x53D7;&#x611F;&#x67D3;&#x7EC6;&#x80DE;&#x7684;&#x53D1;&#x751F;&#x3002;&#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x4F53;&#x76AE;&#x86CB;&#x767D; pp150 (ppUL32) &#x662F;&#x91CD;&#x8981;&#x7684;&#x4EBA;&#x7C7B;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x7684;&#x86CB;&#x767D;&#x8D28;&#x548C;&#x66FE;&#x5EFA;&#x8BAE;&#x5728;&#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x9636;&#x6BB5;&#x53D1;&#x6325;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x8981;&#x8FDB;&#x4E00;&#x6B65;&#x754C;&#x5B9A;&#x5176;&#x4F5C;&#x7528;&#xFF0C;&#x5728;&#x75C5;&#x6BD2;&#x88C5;&#x914D;&#x5E76;&#x67E5;&#x660E;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x671F;&#x95F4;&#x4E0E; pp150 &#x4EA4;&#x4E92;&#x7684;&#x5BBF;&#x4E3B;&#x7EC6;&#x80DE;&#x86CB;&#x767D;&#x8D28;&#xFF0C;&#x6211;&#x4EEC;&#x5229;&#x7528;&#x9175;&#x6BCD;&#x53CC;&#x6742;&#x4EA4;&#x5206;&#x6790;&#x6765;&#x68C0;&#x6D4B; pp150 &#x548C; Bicaudal D1 &#x4E4B;&#x95F4;&#x7684;&#x4EA4;&#x4E92; (BicD1) &#x86CB;&#x767D;&#x60F3;&#x5728;&#x8D29;&#x5356;&#x4EBA;&#x53E3;&#x7684;&#x5206;&#x6CCC;&#x9014;&#x5F84;&#x53D1;&#x6325;&#x4F5C;&#x7528;&#x3002;BicD1 &#x5DF2;&#x77E5;&#x4E0E;&#x590D;&#x6742;&#x7684;&#x808C;&#x7535;&#x673A;&#x548C; Rab6 &#x86CB;&#x767D;&#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#x3002;Pp150 &#x548C; BicD1 &#x4E4B;&#x95F4;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x8BC1;&#x5B9E; coimmunoprecipitation &#x548C;&#x8367;&#x5149;&#x5171;&#x632F;&#x80FD;&#x91CF;&#x8F6C;&#x79FB;&#x3002;&#x77ED;&#x53D1;&#x5939; RNA (shRNA) &#x4E0E; BicD1 &#x7684;&#x67AF;&#x7AED;&#x9020;&#x6210;&#x4EA7;&#x91CF;&#x51CF;&#x5C11;&#x7684;&#x75C5;&#x6BD2;&#x548C;&#x8D29;&#x8FD0;&#x5230;&#x7EC6;&#x80DE;&#x8D28;&#x75C5;&#x6BD2;&#x6027;&#x5927;&#x4F1A;&#x8F66;&#x53A2; (AC) pp150 &#x4E2D;&#x7684;&#x7F3A;&#x9677;&#x800C;&#x4E0D;&#x6539;&#x53D8;&#x8D29;&#x8FD0;&#x5230;&#x7684;&#x53E6;&#x4E00;&#x4E2A;&#x91CD;&#x8981;&#x76AE;&#x86CB;&#x767D;&#x8D28;&#x3001; pp28 &#x6216;&#x75C5;&#x6BD2;&#x7CD6;&#x86CB;&#x767D;&#x590D;&#x6742; gM/gN.&#x4EA4;&#x6D41;BicD1 &#x7684; C &#x603B;&#x7AD9;&#x66FE;&#x88AB;&#x8BC1;&#x660E;&#x53EF;&#x4EA4;&#x4E92;&#x86CB;&#x767D; Rab6&#xFF0C;&#x6697;&#x793A; Rab6 &#x4ECB;&#x5BFC;&#x6C34;&#x75B1;&#x8D29;&#x5356;&#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x6F5C;&#x5728;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x622A;&#x65AD; BicD1 N &#x603B;&#x7AD9;&#x7684;&#x8FC7;&#x5EA6;&#x8868;&#x8FBE;&#x663E;&#x6027;&#x8D1F;&#x7684;&#x65B9;&#x5F0F;&#x884C;&#x4E3A;&#x5E76;&#x5BFC;&#x81F4;&#x4EA4;&#x6D41;&#x548C;&#x4F20;&#x67D3;&#x6027;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x51CF;&#x5C11;&#x4E2D;&#x65AD;&#x3002;&#x6B64;&#x8868;&#x578B;&#x662F;&#x7C7B;&#x4F3C;&#x4E8E; dynamitin &#xFF08;p50&#xFF09;&#xFF0C;&#x89C2;&#x5BDF;&#x5230;&#x4EE5;&#x4E0B;&#x8FC7;&#x91CF;&#x8868;&#x8FBE;&#xFF0C;&#x5E76;&#x63D0;&#x4F9B;&#x8865;&#x5145;&#x8BC1;&#x636E;&#x7684;&#x4EA4;&#x6D41;&#x548C;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x7684;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x4E86;&#x808C;&#x4F9D;&#x8D56;&#x3002;

Bicaudal D1 &#x4F9D;&#x8D56;&#x4EBA;&#x7C7B;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x76AE;&#x86CB;&#x767D;&#x8D28; pp150 &#x5728;&#x75C5;&#x6BD2;&#x611F;&#x67D3;&#x7EC6;&#x80DE;&#x7684;&#x8D29;&#x8FD0;&#x3002;

Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.

Bicaudal D1-dependent trafficking of human cytomegalovirus tegument protein pp150 in virus-infected cells.

Assembl&eacute;e du virion cytom&eacute;galovirus humain (HCMV) se d&eacute;roule dans le noyau et le cytoplasme des cellules infect&eacute;es. Le virion HCMV t&eacute;gument prot&eacute;ine pp150 (ppUL32) est une prot&eacute;ine essentielle de HCMV et a sugg&eacute;r&eacute; de jouer un r&ocirc;le dans la phase cytoplasmique de l&#39;Assembl&eacute;e HCMV. Pour mieux d&eacute;finir son r&ocirc;le dans l&#39;assemblage viral et d&#39;identifier des prot&eacute;ines de cellules h&ocirc;tes au cours de l&#39;assemblage viral interagir avec pp150, nous avons eu recours &agrave; des analyses de double-hybride de levure pour d&eacute;tecter une interaction entre pp150 et Bicaudal D1 (BicD1), une prot&eacute;ine semble jouer un r&ocirc;le dans le trafic au sein de la voie de s&eacute;cr&eacute;tion. BicD1 est connu pour interagir avec le moteur de la dyn&eacute;ine complex et la GTPase Rab6. L&#39;interaction entre pp150 et BicD1 a &eacute;t&eacute; confirm&eacute;e par co-immunopr&eacute;cipitation et fluorescence resonance transfert d&#39;&eacute;nergie. Appauvrissement de la couche de BicD1 avec de l&#39;ARN en &eacute;pingle &agrave; cheveux courts (shARN) caus&eacute; rendement virus une diminution et un d&eacute;faut dans le trafic de pp150 au compartiment cytoplasmique assemblage viral (AC), sans alt&eacute;rer le trafic &agrave; l&#39;AC d&#39;une autre prot&eacute;ine essentielle de t&eacute;gument, pp28 ou la glycoprot&eacute;ine virale gM/GN complexes. La partie C terminale de BicD1 a &eacute;t&eacute; d&eacute;montr&eacute;e pr&eacute;c&eacute;demment pour interagir avec la GTPase Rab6, sugg&eacute;rant un r&ocirc;le potentiel pour Rab6 m&eacute;di&eacute;e par le trafic v&eacute;siculaire dans l&#39;Assembl&eacute;e HCMV. Enfin, la surexpression de l&#39;extr&eacute;mit&eacute; N terminale de BicD1 tronqu&eacute; agit de mani&egrave;re n&eacute;gative dominante et conduit &agrave; l&#39;interruption de l&#39;AC et une diminution dans l&#39;assemblage des virus infectieux. Ce ph&eacute;notype est semblable &agrave; cette surexpression observ&eacute;e suivante de dynamitin (p50) et fourni des preuves suppl&eacute;mentaires que la morphogen&egrave;se de l&#39;AC et virus d&eacute;pendaient de dyn&eacute;ine.

Bicaudal D1-d&eacute;pendant du trafic du cytom&eacute;galovirus humain t&eacute;gument pp150 de prot&eacute;ines dans les cellules infect&eacute;es par le virus.

Menschliche Cytomegalievirus (HCMV) Virion Montage erfolgt im Zellkern und Zytoplasma der infizierten Zellen. Die HCMV Virion charakterisierenden Protein pp150 (ppUL32) ist eine wesentliche Protein des HCMV und eine Rolle in der zytoplasmatischen Phase der HCMV-Versammlung vorgeschlagen worden. Um seine Rolle weiter in virale Baugruppe zu definieren und Host Zelle Proteine zu identifizieren, die Interaktion mit pp150 w&auml;hrend der viralen Montage, wir genutzt Hefe-zwei-Hybrid-Analysen um eine Interaktion zwischen pp150 und Bicaudal D1 zu erkennen (BicD1), ein Protein, eine Rolle in den Handel innerhalb des sekretorischen Weges gedacht. BicD1 ist bekannt, dass mit der komplexen Dynein-Motor und die Rab6 GTPase interagieren. Die Interaktion zwischen pp150 und BicD1 wurde durch Coimmunoprecipitation und Fluoreszenz-Resonanz best&auml;tigt Energietransfer. Ersch&ouml;pfung der BicD1 mit kurzen Haarnadel RNA (ShRNA) verursacht verminderte Virus Ausbeute und einen Defekt im Handel mit pp150 zum zytoplasmatischen virale Versammlung Fach (AC), ohne Ver&auml;nderung der Handel auf die AC ein weiterer wesentlicher charakterisierenden Protein, pp28 oder die komplexe virale Glykoprotein gM/GN Das Carboxyl-Terminus der BicD1 hat sich zuvor gezeigt Interaktion mit der GTPase Rab6, was auf eine m&ouml;gliche Rolle f&uuml;r Rab6-vermittelte vesikul&auml;ren Menschenhandel HCMV-Versammlung. Schlie&szlig;lich eine &Uuml;berexpression von der N-Terminus des abgeschnittenen BicD1 fungiert eine Dominant-negativer Weise und f&uuml;hrt zu St&ouml;rung der AC und eine Abnahme in der Versammlung des ansteckenden Virus. Dieser Ph&auml;notyp war &auml;hnlich wie die beobachteten folgende &Uuml;berexpression des dynamitin (p50) und die Morphogenese der AC und Virus Assembly Dynein abh&auml;ngigen wurden zus&auml;tzliche Beweise vorgelegt.

Bicaudal D1-abh&auml;ngige illegalen Handel mit menschlichen Zytomegalovirus charakterisierenden Protein pp150 in Virus-infizierten Zellen.

Assemblaggio del virione citomegalovirus umano (HCMV) avviene nel nucleo e citoplasma delle cellule infette. L&#39;HCMV virione tegumento proteico pp150 (ppUL32) &egrave; una proteina essenziale di HCMV ed &egrave; stato suggerito di giocare un ruolo nella fase citoplasmatica dell&#39;Assemblea HCMV. Utilizzati per definire ulteriormente il suo ruolo nell&#39;assembly virale e per identificare le proteine delle cellule ospite che interagiscono con pp150 durante l&#39;assemblaggio virale, analisi two-hybrid lievito per rilevare un&#39;interazione tra pp150 e Bicd2 D1 (BicD1), una proteina pensato a giocare un ruolo nel traffico all&#39;interno del percorso secretorio. BicD1 &egrave; noto per interagire con il motore di dineina complesso e il GTPase Rab6. L&#39;interazione tra pp150 e BicD1 &egrave; stata confermata dalla risonanza di fluorescenza e coimmunoprecipitation il trasferimento di energia. Deplezione di BicD1 con short hairpin RNA (shRNA) causato rendimento diminuito virus e un difetto nel traffico di pp150 al compartimento citoplasmatico assemblaggio virale (AC), senza alterare il traffico per l&#39;AC di un&#39;altra proteina essenziale tegumento, pp28 o la complessa glicoproteina virale gM/GN.. C terminale di BicD1 &egrave; stato precedentemente dimostrato di interagire con il GTPase Rab6, suggerendo un ruolo potenziale di traffico vescicolare Rab6-mediata in Assemblea HCMV. Infine, iperespressione del capolinea del tronco BicD1 N agisce in maniera dominante-negativo e porta alla perturbazione dell&#39;AC e una diminuzione nell&#39;Assemblea del virus infettivo. Questo fenotipo &egrave; stato simile a quello osservato sovraespressione seguente di dynamitin (p50) e fornito ulteriori evidenze che morfogenesi dell&#39;Assemblea AC e virus erano dineina dipendente.

BICD2 D1-dipendente dal traffico di citomegalovirus umano tegumento pp150 proteina nelle cellule infettate da virus.

&#x30D2;&#x30C8;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9; (HCMV) &#x30D3;&#x30EA;&#x30AA;&#x30F3; &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x6838;&#x304A;&#x3088;&#x3073;&#x611F;&#x67D3;&#x7D30;&#x80DE;&#x306E;&#x7D30;&#x80DE;&#x8CEA;&#x3067;&#x884C;&#x308F;&#x308C;&#x307E;&#x3059;&#x3002;HCMV &#x30D3;&#x30EA;&#x30AA;&#x30F3; &#x30C6;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x30BF;&#x30F3;&#x30D1;&#x30AF; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; pp150 (ppUL32) HCMV &#x306E;&#x91CD;&#x8981;&#x306A;&#x86CB;&#x767D;&#x8CEA;&#x3067;&#x3042;&#x308A;&#x3001;HCMV &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x7D30;&#x80DE;&#x8CEA;&#x306E;&#x6BB5;&#x968E;&#x3067;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3067;&#x305D;&#x306E;&#x5F79;&#x5272;&#x3092;&#x5B9A;&#x7FA9;&#x3059;&#x308B; pp150 &#x3068;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x4E2D;&#x5BFE;&#x8A71;&#x30DB;&#x30B9;&#x30C8; &#x30BB;&#x30EB;&#x306E;&#x86CB;&#x767D;&#x8CEA;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F; pp150 &#x3068; Bicaudal D1 &#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x9175;&#x6BCD; 2 &#x30CF;&#x30A4;&#x30D6;&#x30EA;&#x30C3;&#x30C9;&#x89E3;&#x6790;&#x6D3B;&#x7528; (BicD1) &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x8003;&#x3048;&#x305F;&#x5185;&#x5206;&#x6CCC;&#x7D4C;&#x8DEF;&#x3092;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x3002;BicD1 &#x30C0;&#x30A4;&#x30CB;&#x30F3; &#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x8907;&#x5408;&#x304A;&#x3088;&#x3073; Rab6 GTPase &#x3068;&#x5BFE;&#x8A71;&#x3059;&#x308B;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;Coimmunoprecipitation &#x3068;&#x86CD;&#x5149;&#x5171;&#x9CF4;&#x306B;&#x3088;&#x308B; pp150 &#x3068; BicD1 &#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x304C;&#x78BA;&#x8A8D;&#x3055;&#x308C;&#x305F;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x4F1D;&#x9054;&#x3002;&#x5225;&#x306E;&#x5FC5;&#x9808;&#x306E;&#x30C6;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x86CB;&#x767D;&#x8CEA;&#x3001;pp28&#x3001;&#x307E;&#x305F;&#x306F;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x7CD6;&#x8907;&#x96D1;&#x306A; gM &#x8205;&#x306E; AC &#x306B;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3092;&#x5909;&#x66F4;&#x3059;&#x308B;&#x3053;&#x3068;&#x306A;&#x304F;&#x304C;&#x6E1B;&#x5C11;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x53CE;&#x91CF;&#x304A;&#x3088;&#x3073; pp150 &#x7D30;&#x80DE;&#x8CEA;&#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA; &#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8; (AC) &#x3078;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3067;&#x6B20;&#x9665; BicD1 &#x77ED;&#x9396;&#x30D8;&#x30A2;&#x30D4;&#x30F3;&#x578B; RNA &#xFF08;shRNA&#xFF09; &#x3068;&#x306E;&#x67AF;&#x6E07;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3057;&#x305F;BicD1 &#x306E; C &#x672B;&#x7AEF;&#x4EE5;&#x524D; GTPase Rab6 &#x3068;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3059;&#x308B; Rab6 &#x3092;&#x4ECB;&#x3057;&#x305F;&#x5C0F;&#x80DE; HCMV &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x3067;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x5F79;&#x5272;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x5207;&#x308A;&#x8A70;&#x3081;&#x3089;&#x308C;&#x305F; BicD1 &#x306E; N &#x672B;&#x7AEF;&#x306E;&#x904E;&#x5270;&#x767A;&#x73FE;&#x306F;&#x30C9;&#x30DF;&#x30CA;&#x30F3;&#x30C8; &#x30CD;&#x30AC;&#x30C6;&#x30A3;&#x30D6;&#x884C;&#x70BA;&#x304C;&#x3001;AC &#x3068;&#x4F1D;&#x67D3;&#x6027;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x6E1B;&#x5C11;&#x306E;&#x4E2D;&#x65AD;&#x306B; &#x3002;&#x3053;&#x306E;&#x8868;&#x73FE;&#x578B; dynamitin (p50) &#x306E;&#x89B3;&#x6E2C;&#x6B21;&#x904E;&#x5270;&#x767A;&#x73FE;&#x306B;&#x4F3C;&#x3066;&#x3044;&#x305F;&#x3057;&#x3001;&#x30C0;&#x30A4;&#x30CB;&#x30F3;&#x304C;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x305F;&#x305D;&#x306E;&#x5F62;&#x614B;&#x5F62;&#x6210; AC &#x304A;&#x3088;&#x3073;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x8FFD;&#x52A0;&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

Bicaudal D1 &#x306B;&#x4F9D;&#x5B58;&#x3059;&#x308B;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x611F;&#x67D3;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x30D2;&#x30C8;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED; &#x30C6;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x30BF;&#x30F3;&#x30D1;&#x30AF; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; pp150 &#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x3002;

&#xC778;&#xAC04;&#xC758; &#xC138;&#xD3EC; (HCMV) virion &#xC5B4;&#xC148;&#xBE14;&#xB9AC; &#xD575; &#xBC0F; &#xAC10;&#xC5FC; &#xB41C; &#xC138;&#xD3EC;&#xC758; &#xC138;&#xD3EC;&#xC9C8;&#xC5D0;&#xC11C; &#xC77C;&#xC5B4;&#xB09C;&#xB2E4;. HCMV virion tegument &#xB2E8;&#xBC31;&#xC9C8; pp150 (ppUL32) HCMV&#xC758; &#xD544;&#xC218;&#xC801;&#xC778; &#xB2E8;&#xBC31;&#xC9C8; &#xC774;&#xBA70; HCMV &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC758; &#xC138;&#xD3EC;&#xC9C8; &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xC6B0;&#xB9AC; &#xD6A8; &#xBAA8; 2 &#xC7A1;&#xC885; &#xBD84;&#xC11D; pp150 Bicaudal D1 &#xC0AC;&#xC774; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xAC10;&#xC9C0; &#xD558;&#xB294; &#xD65C;&#xC6A9; &#xB354; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0;&#xC758; &#xC5ED;&#xD560;&#xC744; &#xC815;&#xC758; &#xD558; &#xACE0; &#xC2DD;&#xBCC4; pp150 &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xC870;&#xB9BD; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC; &#xB2E8;&#xBC31;&#xC9C8; (BicD1) &#xB2E8;&#xBC31;&#xC9C8; &#xBD84; &#xD1B5;&#xB85C; &#xB0B4;&#xC5D0;&#xC11C; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xC5ED;&#xD560;&#xC744; &#xD560; &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01; &#xD569;&#xB2C8;&#xB2E4;. Bicd1&#xB294; &#xBCF5;&#xC7A1; &#xD55C; &#xD68C; &#xBAA8;&#xD130;&#xC640; Rab6 GTPase &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC838; &#xC788;&#xB2E4;. Pp150&#xC640; BicD1 &#xAC04;&#xC758; &#xC0C1;&#xD638; &#xC791;&#xC6A9; coimmunoprecipitation &#xBC0F; &#xD615;&#xAD11; &#xACF5;&#xBA85;&#xC5D0; &#xC758;&#xD574; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xB2E4; &#xC5D0;&#xB108;&#xC9C0; &#xC804;&#xC1A1;. &#xC9E7;&#xC740; &#xD5E4;&#xC5B4;&#xD540; RNA (shRNA)&#xC640; Bicd1&#xC758; &#xACE0;&#xAC08; &#xB2E4;&#xB978; &#xD544;&#xC218; tegument &#xB2E8;&#xBC31;&#xC9C8;, pp28, &#xB610;&#xB294; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xB2F9;&#xB2E8;&#xBC31;&#xC9C8; &#xBCF5;&#xC7A1; &#xD55C; gM/Gn.&#xC758; AC&#xC5D0; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xB97C; &#xBCC0;&#xACBD; &#xD558;&#xC9C0; &#xC54A;&#xACE0; &#xAC10;&#xC18C; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBC0F; &#xC138;&#xD3EC;&#xC9C8; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC5B4;&#xC148;&#xBE14;&#xB9AC; &#xAD6C;&#xD68D; (AC)&#xC5D0; pp150&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC5D0; &#xACB0;&#xD568; &#xBC1C;&#xC0DD; Bicd1&#xC758; C &#xB9D0;&#xB2E8; &#xC774;&#xC804;&#xC5D0; &#xBCF4;&#xC5EC;&#xC654;&#xB2E4; GTPase Rab6 &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD558;&#xB294; HCMV &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC758; &#xAE30;&#xACF5;&#xC744; &#xB9E4;&#xB9E4; Rab6 &#xC911;&#xC7AC;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xC81C;&#xC548;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C; &#xC798;&#xB9B0;&#xB41C; Bicd1&#xC758; N &#xB9D0;&#xB2E8;&#xC758; overexpression &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xBD80;&#xC815;&#xC801;&#xC778; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xB0B4;&#xC5D0;&#xC11C; &#xC791;&#xB3D9; &#xD558; &#xACE0; AC &#xBC0F; &#xC804;&#xC5FC; &#xC131; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0; &#xC788;&#xB294; &#xAC10;&#xC18C;&#xC758; &#xC911;&#xB2E8;&#xC5D0; &#xC774;&#xB974;&#xAC8C;. &#xC774; &#xD615; dynamitin (p50)&#xC758; &#xAD00;&#xCC30; &#xB41C; &#xB2E4;&#xC74C; overexpression &#xADF8;&#xC640; &#xBE44;&#xC2B7;&#xD55C; &#xACE0; &#xCD94;&#xAC00; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xADF8; morphogenesis AC &#xBC0F; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC758; &#xD68C; &#xC758;&#xC874; &#xD588;&#xB2E4;.

Bicaudal D1 &#xC885;&#xC18D; &#xC778;&#xAC04;&#xC758; &#xC138;&#xD3EC; tegument &#xB2E8;&#xBC31;&#xC9C8; pp150 &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC5D0; &#xAC10;&#xC5FC; &#xB41C; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xC758; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;.

Asamblea de viri&oacute;n humana del citomegalovirus (HCMV) ocurre en el n&uacute;cleo y el citoplasma de las c&eacute;lulas infectadas. El HCMV virion tegumento prote&iacute;na pp150 (ppUL32) es una prote&iacute;na esencial de HCMV y ha sugerido a desempe&ntilde;ar un papel en la fase citopl&aacute;smica de Asamblea HCMV. Para definir su papel en el ensamblaje viral e identificar prote&iacute;nas de la c&eacute;lula hu&eacute;sped que interact&uacute;an con pp150 durante el ensamblaje viral, se utilizaron el an&aacute;lisis de dos h&iacute;bridos de levadura para detectar una interacci&oacute;n entre pp150 y Bicaudal D1 (BicD1), una prote&iacute;na se piensa para desempe&ntilde;ar un papel en el tr&aacute;fico en la v&iacute;a secretora. BicD1 se sabe que interact&uacute;an con el motor de la dine&iacute;na complejo y la GTPasa de Rab6. La interacci&oacute;n entre pp150 y BicD1 fue confirmada por coimmunoprecipitation y fluorescencia de resonancia la transferencia de energ&iacute;a. Agotamiento de BicD1 con short hairpin RNA (shRNA) causada virus menor rendimiento y un defecto en el tr&aacute;fico de pp150 del compartimiento de ensamblaje viral citopl&aacute;smica (AC), sin alterar el tr&aacute;fico a la AC de otra prote&iacute;na esencial del tegumento, pp28 o el complejo de la glicoprote&iacute;na viral gM/GN La terminal C de BicD1 ha demostrado previamente para interactuar con el GTPase Rab6, sugiriendo un papel potencial para el tr&aacute;fico vesicular Rab6-mediada en Asamblea HCMV. Finalmente, la sobreexpresi&oacute;n de N terminal de BicD1 truncado act&uacute;a de una manera dominante negativo y conduce a la interrupci&oacute;n de la AC y una disminuci&oacute;n en la Asamblea de virus infeccioso. Este fenotipo era similar a la que observado siguiente la sobreexpresi&oacute;n de dynamitin (p50) y proporciona evidencia adicional morfog&eacute;nesis de la Asamblea de AC y virus eran dependientes dine&iacute;na.

Bicaudal D1-dependiente trata de citomegalovirus humano tegumento prote&iacute;na pp150 en c&eacute;lulas infectadas por virus.

En humanos-citomegalovirus (HCMV)-c&eacute;lulas infectadas, la localizaci&oacute;n de la prote&iacute;na viral pp150 del compartimiento de la Asamblea de virus (AC) depende de su interacci&oacute;n directa con la prote&iacute;na celular Bicaudal D1 a trav&eacute;s de un mecanismo dependiente de microt&uacute;bulos y dine&iacute;na. Descubrimos que el peque&ntilde;o que GTPase Rab6 tambi&eacute;n interact&uacute;a indirectamente con pp150 a trav&eacute;s de su interacci&oacute;n con Bicaudal D1. Inhibici&oacute;n de la actividad Rab6 en c&eacute;lulas infectadas HCMV hab&iacute;a interrumpido el tr&aacute;fico intracelular de pp150, reduciendo significativamente la producci&oacute;n de virus infeccioso sin afectar a la formaci&oacute;n de la CA, reclaman una funci&oacute;n importante para este celular GTPasa en la localizaci&oacute;n intracelular de pp150 durante el ensamblaje del virus.

Un papel para el peque&ntilde;o GTPase Rab6 en Asamblea del citomegalovirus humano.

&#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2; (HCMV)-&#x53D7;&#x611F;&#x67D3;&#x7684;&#x7EC6;&#x80DE;&#xFF0C;&#x672C;&#x5730;&#x5316;&#x7684;&#x75C5;&#x6BD2;&#x86CB;&#x767D;&#x8D28; pp150 &#x5230;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x8F66;&#x53A2; (AC) &#x662F;&#x4F9D;&#x8D56;&#x4E0E;&#x7EC6;&#x80DE;&#x86CB;&#x767D; Bicaudal D1 &#x808C;&#x548C;&#x5FAE;&#x7BA1;&#x4F9D;&#x8D56;&#x673A;&#x5236;&#x901A;&#x8FC7;&#x5176;&#x76F4;&#x63A5;&#x4EA4;&#x4E92;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x7684;&#x5C0F;&#x86CB;&#x767D; Rab6 &#x4E5F;&#x95F4;&#x63A5;&#x4EA4;&#x4E92; pp150 &#x901A;&#x8FC7;&#x5176;&#x4E0E; Bicaudal D1 &#x7684;&#x4E92;&#x52A8;&#x3002;Rab6 &#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x611F;&#x67D3;&#x7684;&#x7EC6;&#x80DE;&#x6D3B;&#x6027;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x4E2D;&#x65AD;&#x80DE;&#x5185;&#x8D29;&#x5356; pp150&#xFF0C;&#x5927;&#x5927;&#x51CF;&#x5C11;&#x4F20;&#x67D3;&#x6027;&#x75C5;&#x6BD2;&#x751F;&#x4EA7;&#x6CA1;&#x6709;&#x5F71;&#x54CD;&#x5F62;&#x6210;&#x7684;&#x4EA4;&#x6D41;&#xFF0C;&#x4E3A;&#x6B64;&#x7EC6;&#x80DE;&#x86CB;&#x767D; pp150 &#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x671F;&#x95F4;&#x80DE;&#x5185;&#x5B9A;&#x4F4D;&#x4E2D;&#x4E89;&#x8BBA;&#x7684;&#x4E00;&#x9879;&#x91CD;&#x8981;&#x804C;&#x80FD;&#x3002;

&#x4EBA;&#x5DE8;&#x7EC6;&#x80DE;&#x75C5;&#x6BD2;&#x5927;&#x4F1A;&#x4E2D;&#x7684;&#x5C0F;&#x86CB;&#x767D; Rab6 &#x7684;&#x4F5C;&#x7528;&#x3002;

In human-cytomegalovirus (HCMV)-infected cells, the localization of the viral protein pp150 to the virus assembly compartment (AC) is dependent on its direct interaction with the cellular protein Bicaudal D1 through a dynein- and microtubule-dependent mechanism. We found that the small GTPase Rab6 also interacts indirectly with pp150 through its interaction with Bicaudal D1. Inhibition of Rab6 activity in HCMV-infected cells interrupted the intracellular trafficking of pp150, significantly reducing infectious virus production without affecting the formation of the AC, arguing for an important function for this cellular GTPase in the intracellular localization of pp150 during virus assembly.

A role for the small GTPase Rab6 in assembly of human cytomegalovirus.

En homme-cytom&eacute;galovirus (HCMV)-cellules infect&eacute;es, la localisation de la prot&eacute;ine virale de pp150 dans le compartiment de l&#39;Assembl&eacute;e de virus (AC) d&eacute;pend de l&#39;interaction directe avec la prot&eacute;ine cellulaire Bicaudal D1 par un m&eacute;canisme d&eacute;pendant de dyn&eacute;ine et microtubules. Nous avons constat&eacute; que la petite que GTPase Rab6 interagit aussi indirectement avec pp150 gr&acirc;ce &agrave; son interaction avec Bicaudal D1. Inhibition de l&#39;activit&eacute; dans les cellules infect&eacute;es par HCMV Rab6 interrompu le trafic intracellulaire de pp150, r&eacute;duisant consid&eacute;rablement la production de virus infectieux sans affecter la formation du Comit&eacute; d&#39;audit, plaidant pour une fonction importante pour cette GTPase cellulaire dans la localisation intracellulaire de pp150 lors de l&#39;assemblage du virus.

Un r&ocirc;le pour le petit GTPase Rab6 dans l&#39;Assembl&eacute;e du cytom&eacute;galovirus humain.

In Mensch-Cytomegalievirus (HCMV)-infizierte Zellen, die Sprachunterst&uuml;tzung f&uuml;r das virale Protein pp150 zum Virus Versammlung Fach (AC) ist die direkte Interaktion mit dem zellularen Protein Bicaudal D1 durch Dynein - und Microtubule-abh&auml;ngigen Mechanismus abh&auml;ngig. Wir fanden, dass die kleinen, die GTPase Rab6 auch indirekt mit pp150 durch seine Interaktion mit Bicaudal D1 interagiert. Hemmung der Rab6 Aktivit&auml;t in HCMV-infizierten Zellen unterbrochen, der intrazellul&auml;ren Menschenhandel pp150, infekti&ouml;se Virus-Produktion deutlich zu verringern, ohne die Bildung von AC, als Argument f&uuml;r eine wichtige Funktion f&uuml;r diese zellul&auml;re GTPase in die intrazellul&auml;re Lokalisation der pp150 bei der Virus-Montage.

Eine Rolle f&uuml;r die kleine GTPase Rab6 in Versammlung der menschlichen Zytomegalovirus.

Nell&#39;uomo-citomegalovirus (HCMV)-cellule infette, la localizzazione della proteina virale pp150 per il vano di montaggio virus (AC) dipende dalla sua diretta interazione con la proteina cellulare Bicd2 D1 attraverso un meccanismo di dineina e microtubule-dipendente. Abbiamo scoperto che il piccolo che GTPase Rab6 interagisce anche indirettamente con pp150 attraverso la sua interazione con Bicd2 D1. Inibizione dell&#39;attivit&agrave; Rab6 in cellule infettate da HCMV interrotto il traffico intracellulare di pp150, riducendo significativamente la produzione di virus infettivo senza alterare la formazione di AC, sostenendo un&#39;importante funzione per questo cellulare GTPase nella localizzazione intracellulare di pp150 durante l&#39;assemblaggio del virus.

Un ruolo per il piccolo GTPase Rab6 Assemblea del citomegalovirus umano.

&#x4EBA;&#x9593;-&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9; (HCMV) &#x3067;-&#x611F;&#x67D3;&#x7D30;&#x80DE;&#x3001;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA; &#x30B3;&#x30F3;&#x30D1;&#x30FC;&#x30C8;&#x30E1;&#x30F3;&#x30C8; (AC) &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x86CB;&#x767D;&#x8CEA; pp150 &#x306E;&#x5C40;&#x5728;&#x306F;&#x7D30;&#x80DE;&#x306E;&#x86CB;&#x767D;&#x8CEA; Bicaudal D1 &#x30C0;&#x30A4;&#x30CB;&#x30F3;-&#x5FAE;&#x5C0F;&#x7BA1;&#x4F9D;&#x5B58;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x901A;&#x3058;&#x3066;&#x306E;&#x76F4;&#x63A5;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F; gtp &#x30A2;&#x30FC;&#x30BC; Rab6 &#x3082;&#x76F4;&#x63A5; pp150 Bicaudal D1 &#x3068;&#x306E;&#x4EA4;&#x6D41;&#x306B;&#x3088;&#x308B;&#x5BFE;&#x8A71;&#x3057;&#x306A;&#x3044;&#x5C0F;&#x3055;&#x306A;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;HCMV &#x611F;&#x67D3;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B; Rab6 &#x6D3B;&#x6027;&#x306E;&#x963B;&#x5BB3;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x5185;&#x306E;&#x4EBA;&#x8EAB;&#x58F2;&#x8CB7;&#x306E; pp150 &#x3053;&#x306E;&#x30BB;&#x30EB;&#x30E9;&#x30FC; GTPase pp150 &#x306E;&#x7D30;&#x80DE;&#x5185;&#x5C40;&#x5728;&#x30A6;&#x30A4;&#x30EB;&#x30B9; &#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x4E2D;&#x306E;&#x91CD;&#x8981;&#x306A;&#x6A5F;&#x80FD;&#x3092;&#x4E3B;&#x5F35;&#x3001;AC &#x306E;&#x5F62;&#x6210;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x305A;&#x3001;&#x4F1D;&#x67D3;&#x6027;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x751F;&#x7523;&#x3092;&#x5927;&#x5E45;&#x306B;&#x524A;&#x6E1B;&#x3001;&#x4E2D;&#x65AD;&#x3002;

&#x5C0F;&#x3055;&#x306A; GTPase Rab6 &#x30D2;&#x30C8;&#x30B5;&#x30A4;&#x30C8;&#x30E1;&#x30AC;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x5185;&#x306E;&#x30ED;&#x30FC;&#x30EB;&#x3002;

&#xC778;&#xAC04; &#xC138;&#xD3EC; (HCMV)&#xC5D0;&#xC11C;-&#xAC10;&#xC5FC; &#xB41C; &#xC138;&#xD3EC;, &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC5B4;&#xC148;&#xBE14;&#xB9AC; &#xAD6C;&#xD68D; (AC)&#xC5D0; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xB2E8;&#xBC31;&#xC9C8; pp150 &#xC9C0;&#xC5ED;&#xD654; &#xD68C; &#xBC0F; microtubule &#xC885;&#xC18D; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD1B5;&#xD574; &#xC138;&#xD3EC;&#xC9C8; &#xB2E8;&#xBC31;&#xC9C8; Bicaudal d 1&#xC640;&#xC758; &#xC9C1;&#xC811; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; GTPase Rab6 &#xB610;&#xD55C; &#xD558;&#xC9C0; &#xC9C1;&#xC811; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC758; &#xC0C1;&#xD638; Bicaudal d 1&#xC744; &#xD1B5;&#xD574; pp150 &#xC791;&#xC740; &#xBC1C;&#xACAC;. HCMV&#xC5D0; &#xAC10;&#xC5FC; &#xB41C; &#xC138;&#xD3EC;&#xC5D0; Rab6 &#xD65C;&#xB3D9;&#xC758; &#xC800;&#xD574; pp150, &#xD06C;&#xAC8C;&#xC774; &#xC140;&#xB8F0;&#xB7EC; GTPase &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC870;&#xB9BD; &#xD558;&#xB294; &#xB3D9;&#xC548; pp150 &#xC138;&#xD3EC;&#xB0B4; &#xC9C0;&#xC5ED;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xAE30;&#xB2A5;&#xC5D0; &#xB300; &#xD55C; &#xB17C;&#xC7C1; AC&#xC758; &#xD615;&#xC131;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xACE0; &#xC804;&#xC5FC; &#xC131; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC0DD;&#xC0B0;&#xC744; &#xAC10;&#xC18C;&#xC758; &#xC138;&#xD3EC;&#xB0B4; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4; &#xC911;&#xB2E8;.

&#xC778;&#xAC04; &#xC138;&#xD3EC;&#xC758; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0; &#xC788;&#xB294; &#xC791;&#xC740; GTPase rab6&#xC5D0; &#xB300; &#xD55C; &#xC5ED;&#xD560;.

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.

Rift Valley fever (RVF) is endemic to sub-Saharan Africa, and has spread into Madagascar, Egypt, Saudi Arabia, and Yemen. Rift Valley fever virus (RVFV) of the family Bunyaviridae, genus Phlebovirus causes hemorrhagic fever, neurological disorders or blindness in humans, and high rate abortion and fetal malformation in ruminants. RVFV is classified as a Category A Priority pathogen and overlap select agent by CDC/USDA due to its potential impact on public health and agriculture. There is a gap in the safety and immunogenicity in traditional RVF vaccines; the formalin-inactivated RVFV vaccine TSI-GSD-200 requires three doses for protection, and the live-attenuated Smithburn vaccine has a risk to cause abortion and fetal malformation in pregnant ruminants. In this review, problems of traditional vaccines and the safety and efficacy of recently reported novel RVF candidate vaccines including subunit vaccines, virus vector, and replicons are discussed.

Novel approaches to develop Rift Valley fever vaccines.

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.

Sabarish V. Indran

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