amplification-next-generation-sequencing-genomic-dna-mapping

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Dana-Farber Cancer Institute

Merck's MK-0518, known as raltegravir, has recently become the first FDA-approved HIV-1 integrase (IN) inhibitor and has since risen to blockbuster drug status. Much research has in turn been conducted over the last few years aimed at recreating but optimizing the compound's interactions with the protein. Resulting me-too drugs have shown favorable pharmacokinetic properties and appear drug-like but, as expected, most have a highly similar interaction with IN to that of raltegravir. We propose that, based upon conclusions drawn from our docking studies illustrated herein, most of these me-too MK-0518 analogues may experience a low success rate against raltegravir-resistant HIV strains. As HIV has a very high mutational competence, the development of drugs with new mechanisms of inhibitory action and/or new active substituents may be a more successful route to take in the development of second- and third-generation IN inhibitors.

Raltegravir, elvitegravir, and metoogravir: the birth of "me-too" HIV-1 integrase inhibitors.

In recent years, HIV-1 integrase (IN) has become an attractive target for designing antiretroviral agents. The first IN inhibitor approved for clinical use, raltegravir, has validated the pharmacological viability of IN inhibitors and signals the advent of a new generation of antiretroviral drugs. The development of raltegravir and other successful lead IN inhibitors has also influenced the IN inhibitor design strategy. This has led to the identification of several potent inhibitors in these last two years. Further, an increased understanding of IN structural biology has opened up novel approaches to inhibiting IN, such as targeting its multimerization or interaction with cellular cofactors. This review covers recent developments in the field of IN inhibitor design from 2007 to 2008.  

HIV-1 integrase inhibitors: 2007-2008 update.

Hiv-1 (IN) &#x662F;&#x5F53;&#x4EE3;&#x6297;&#x9006;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x836F;&#x7269;&#x8BBE;&#x8BA1;&#x7814;&#x7A76;&#x4E2D;&#x5FC3;&#x7684;&#x4E00;&#x9879;&#x91CD;&#x8981;&#x6307;&#x6807;&#x3002;&#x4ECE;&#x5386;&#x53F2;&#x4E0A;&#x770B;&#xFF0C;&#x706D;&#x6D3B;&#x9176;&#x7684;&#x52AA;&#x529B;&#x628A;&#x91CD;&#x70B9;&#x653E;&#x5728;&#x963B;&#x6B62;&#x5176;&#x6D3B;&#x52A8;&#x7F51;&#x7AD9;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x5F88;&#x660E;&#x663E;&#x7684;&#x53D8;&#x6784;&#x6291;&#x5236;&#x5242;&#x7684;&#x65B0;&#x7C7B;&#x5C06;&#x9700;&#x8981;&#x63A8;&#x52A8;&#x6297;&#x53CD;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x7684;&#x75C5;&#x6BD2;&#x6BD2;&#x682A;&#x6297;&#x6027;&#x5F53;&#x4EE3;&#x51FA;&#x73B0;&#x5916;&#x5730;&#x4E34;&#x5E8A;&#x5E38;&#x7528;&#x836F;&#x7269;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x4E2D;&#x6211;&#x4EEC;&#x6709;&#x7279;&#x70B9;&#x7D27;&#x5BC6;&#x7684;&#x7F51;&#x7EDC;&#x7684;&#x6B8B;&#x7559;&#x7269;&#xFF0C;&#x8FDC;&#x4ECE;&#x6D3B;&#x52A8;&#x7F51;&#x7AD9;&#xFF0C;&#x4F5C;&#x4E3A;&#x91CD;&#x8981;&#x7684;&#x5F3A;&#x5236;&#x6027; multimerization &#x7684;&#x9176;&#x548C;&#x4F5C;&#x4E3A;&#x4E00;&#x4E2A;&#x6574;&#x4F53;&#x7684;&#x75C5;&#x6BD2;&#x590D;&#x5236;&#x4E2D;&#x7684;&#x91CD;&#x8981;&#x6027;&#x3002;&#x5177;&#x4F53;&#x800C;&#x8A00;&#xFF0C;&#x6211;&#x4EEC;&#x5DF2;&#x786E;&#x5B9A;&#x7684;&#x516D;&#x4E2A;&#x6B8B;&#x7559;&#x5728;&#x4E2D;&#x4E8C;&#x805A;&#x63A5;&#x53E3;&#xFF0C;&#x7EC4;&#x6210;&#x4E00;&#x4E2A;&#x56DB;&#x7EA7;&#x82B3;&#x9999;&#x4E92;&#x52A8;&#xFF0C;&#x4E24;&#x4FA7;&#x662F;&#x4E24;&#x4E2A;&#x76D0;&#x6865;&#x6881;&#x3001; &#x9AD8;&#x5EA6;&#x5BF9;&#x79F0;&#x7684;&#x533A;&#x57DF;&#x5185;&#x914D;&#x7F6E;&#x5927;&#x5927;&#x6709;&#x52A9;&#x4E8E;&#x6B63;&#x786E;&#x827E;&#x6ECB;&#x75C5;&#x6BD2; 1 &#x590D;&#x5236;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x5229;&#x7528;&#x4E00;&#x79CD;&#x5B9A;&#x91CF;&#x53D1;&#x5149;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#xFF0C;&#x9F50;&#x805A;&#x5728;&#x5BA1;&#x67E5;&#x5E76;&#x5DF2;&#x786E;&#x5B9A;&#x662F;&#x6709;&#x975E;&#x5E38;&#x4F4E;&#x5BB9;&#x5DEE;&#x4E3A;&#x6C28;&#x57FA;&#x9178;&#x66FF;&#x6362;&#x6B64;&#x533A;&#x57DF;&#x6CBF;&#x3002;&#x5728; multimerization&#xFF0C;&#x5728;&#x5904;&#x4E8E;&#x975E;&#x6D3B;&#x52A8;&#x72B6;&#x6001;&#x7684;&#x75C5;&#x6BD2;&#x9176;&#x548C;&#x975E;&#x590D;&#x5236;&#x75C5;&#x6BD2;&#x5BFC;&#x81F4;&#x4EA7;&#x751F;&#x8D1F;&#x9762;&#x5F71;&#x54CD;&#x751A;&#x81F3;&#x4FDD;&#x5B88;&#x6B8B;&#x7559;&#x7684;&#x66FF;&#x6362;&#x3002;&#x6211;&#x4EEC;&#x5DF2;&#x7ECF;&#x8868;&#x660E;&#x7684;&#x7279;&#x70B9;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#xFF0C;&#x8BE5;&#x5BF9;&#x79F0;&#x4E8C;&#x805A;&#x754C;&#x9762;&#x533A;&#x57DF;&#x6C28;&#x57FA;&#x9178;&#x53D8;&#x5316;&#x975E;&#x5E38;&#x4F4E;&#x5BB9;&#x5DEE;&#xFF0C;&#x56E0;&#x6B64;&#x6B64;&#x5904;&#x8BE6;&#x7EC6;&#x4ECB;&#x7ECD;&#x7684;&#x6C28;&#x57FA;&#x9178;&#x7F51;&#x7EDC;&#x4E3A;&#x76EE;&#x6807;&#x7684;&#x836F;&#x7269;&#x53EF;&#x4EE5;&#x9884;&#x8BA1;&#x4EA7;&#x91CF;&#x5927;&#x5927;&#x51CF;&#x5C11;&#x7684;&#x6B21;&#x6570;&#x76F8;&#x6BD4;&#xFF0C;&#x5F53;&#x4EE3;&#x4E34;&#x5E8A;&#x8BC4;&#x4F30;&#x6297;&#x9006;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x836F;&#x7269;&#x8010;&#x836F;&#x7684;&#x8F6C;&#x4E49;&#x7A81;&#x53D8;&#x3002;

&#x5BF9;&#x79F0;&#x533A;&#x57DF;&#x7684; Hiv-1 &#x4E8C;&#x805A;&#x63A5;&#x53E3;&#x662F;&#x75C5;&#x6BD2;&#x590D;&#x5236;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x3002;

HIV-1 integrase (IN) is an important target for contemporary antiretroviral drug design research. Historically, efforts at inactivating the enzyme have focused upon blocking its active site. However, it has become apparent that new classes of allosteric inhibitors will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs. In this study we have characterized the importance of a close network of IN residues, distant from the active site, as important for the obligatory multimerization of the enzyme and viral replication as a whole. Specifically, we have determined that the configuration of six residues within a highly symmetrical region at the IN dimerization interface, composed of a four-tiered aromatic interaction flanked by two salt bridges, significantly contributes to proper HIV-1 replication. Additionally, we have utilized a quantitative luminescence assay to examine IN oligomerization and have determined that there is a very low tolerance for amino acid substitutions along this region. Even conservative residue substitutions negatively impacted IN multimerization, resulting in an inactive viral enzyme and a non-replicative virus. We have shown that there is a very low tolerance for amino acid variation at the symmetrical dimeric interface region characterized in this study, and therefore drugs designed to target the amino acid network detailed here could be expected to yield a significantly reduced number of drug-resistant escape mutations compared to contemporary clinically-evaluated antiretrovirals.

A Symmetric Region of the HIV-1 Integrase Dimerization Interface Is Essential for Viral Replication.

Int&eacute;grase du VIH-1 (IN) est une cible importante pour la recherche en design m&eacute;dicament antir&eacute;troviral contemporain. Historiquement, les efforts &agrave; l&#39;inactivation de l&#39;enzyme ont port&eacute; sur le blocage de son site actif. Cependant, il est devenu apparent que nouvelles classes d&#39;inhibiteurs allost&eacute;riques seront n&eacute;cessaires pour faire avancer le domaine de l&#39;antir&eacute;troviral l&#39;&eacute;mergence de souches virales r&eacute;sistantes &agrave; la cr&eacute;ation contemporaine cliniquement utilis&eacute;es dans les m&eacute;dicaments. Dans cette &eacute;tude, nous avons caract&eacute;ris&eacute; l&#39;importance d&#39;un r&eacute;seau serr&eacute; de r&eacute;sidus, &eacute;loign&eacute;s du site actif, aussi important que la multimerization obligatoire de l&#39;enzyme et la r&eacute;plication virale dans son ensemble. Plus pr&eacute;cis&eacute;ment, nous avons d&eacute;termin&eacute; que la configuration de six r&eacute;sidus dans une r&eacute;gion hautement sym&eacute;trique &agrave; l&#39;interface de dim&eacute;risation IN, compos&eacute; d&#39;une interaction aromatique &agrave; quatre niveaux, flanqu&eacute;e de deux ponts salins, contribue de mani&egrave;re significative &agrave; la bonne r&eacute;plication du VIH-1. En outre, nous avons utilis&eacute; un dosage quantitatif de luminescence pour examiner dans l&#39;oligom&eacute;risation et ont d&eacute;termin&eacute; qu&#39;il y a une tr&egrave;s faible tol&eacute;rance pour les substitutions d&#39;acides amin&eacute;s le long de cette r&eacute;gion. Les substitutions de r&eacute;sidus m&ecirc;me conservateur n&eacute;gativement dans multimerization, ce qui entra&icirc;ne une enzyme virale inactive et un virus non r&eacute;plicatif. Nous avons montr&eacute; qu&#39;il y a une tol&eacute;rance tr&egrave;s faible variation de l&#39;acide amin&eacute; &agrave; la r&eacute;gion d&#39;interface dim&egrave;re sym&eacute;trique caract&eacute;ris&eacute;e dans cette &eacute;tude, et donc m&eacute;dicaments con&ccedil;us pour cibler le r&eacute;seau acides amin&eacute;s d&eacute;taill&eacute; ici risquerait de c&eacute;der un nombre significativement r&eacute;duit de mutations de pharmacor&eacute;sistant &eacute;vasion compar&eacute;e aux antir&eacute;troviraux d&#39;&eacute;valu&eacute; cliniquement contemporain.

Une r&eacute;gion sym&eacute;trique de l&#39;Interface de dim&eacute;risation d&#39;int&eacute;grase du VIH-1 est essentielle &agrave; la r&eacute;plication virale.

HIV-1 Integrase (IN) ist ein wichtiges Ziel f&uuml;r zeitgen&ouml;ssische antiretroviralen Medikament Designforschung. Historisch, konzentrieren sich die Bem&uuml;hungen um das Enzym zu inaktivieren auf seine aktive Website blockieren. Jedoch ist es deutlich geworden, dass neue Klassen von allosterische Inhibitoren auf dem Gebiet der antiretroviralen im Hinblick auf die Entstehung der Virenst&auml;mme, die resistent gegen zeitgen&ouml;ssische voraus sind klinisch IN Drogen verwendet. In dieser Studie haben wir die Bedeutung der ein dichtes Netz von R&uuml;ckst&auml;nde, weit entfernt von der aktiven Site, als wichtig f&uuml;r die obligatorische Multimerization des Enzyms und der Virusreplikation als Ganzes gekennzeichnet. Insbesondere haben wir festgestellt, dass die Konfiguration von sechs R&uuml;ckst&auml;nde innerhalb einer sehr symmetrischen Region an der IN Dimerisierung Schnittstelle, bestehend aus einer vier-Ebenen aromatische Interaktion, flankiert von zwei Salz Br&uuml;cken, signifikant zur richtigen HIV-1-Replikation beitr&auml;gt. Dar&uuml;ber hinaus haben wir eine quantitative Lumineszenz-Assays untersuchen IN Oligomerisierung und haben festgestellt, dass eine sehr geringe Toleranz f&uuml;r Aminos&auml;ure-Substitution entlang dieser Region genutzt. Selbst konservative R&uuml;ckstand Substitutionen negativ IN Multimerization, was ein inaktiver virale Enzym und ein nicht-replicative Virus. Wir haben gezeigt, dass es eine sehr geringe Toleranz f&uuml;r Aminos&auml;ure-Variation an die symmetrische dimeren Schnittstelle-Region, wo in dieser Studie, und daher Medikamente entwickelt, um das Aminos&auml;ure-Netzwerk hier gezielt zu deutlich reduzierten verschiedener medikamentenresistente Escape Mutationen im Vergleich zu zeitgen&ouml;ssischen klinisch evaluiert antiretrovirale Ertrag erwartet werden k&ouml;nnte.

Eine symmetrische Region der HIV-1 Integrase Dimerisierung Schnittstelle ist wesentlich f&uuml;r die virale Replikation.

Integrasi di HIV-1 (IN) sono un obiettivo importante per la ricerca di design farmaco antiretrovirale contemporanea. Storicamente, gli sforzi all&#39;inattivazione dell&#39;enzima sono concentrati su blocca il sito attivo. Tuttavia, &egrave; diventato apparente che nuove classi di inibitori allosterici saranno necessarie per far avanzare il campo antiretrovirale alla luce l&#39;emergere di ceppi virali resistenti al contemporaneo clinicamente utilizzato IN farmaci. In questo studio noi abbiamo caratterizzato l&#39;importanza di una fitta rete di IN residui, distanti dal sito attivo, come importante per la multimerization obbligatoria dell&#39;enzima e replicazione virale nel suo complesso. In particolare, abbiamo determinato che la configurazione di sei residui all&#39;interno di una regione altamente simmetrica all&#39;interfaccia IN dimerizzazione, composto di un&#39;interazione aromatica a quattro livelli affiancata da due ponti sale, contribuisce significativamente alla corretta replicazione dell&#39;HIV-1. Inoltre, abbiamo utilizzato un dosaggio quantitativo luminescenza per esaminare IN oligomerizzazione ed hanno deciso che non c&#39;&egrave; una tolleranza molto bassa per le sostituzioni di aminoacidi lungo questa regione. Le sostituzioni dei residui anche conservatore influenzato negativamente IN multimerization, risultante in un inattivo enzima virale e un virus non replicativa. Abbiamo dimostrato che c&#39;&egrave; una tolleranza molto bassa per variazione dell&#39;amminoacido alla regione di interfaccia dimerica simmetrico caratterizzata in questo studio, e pertanto farmaci progettati per indirizzare l&#39;amminoacido rete dettagliata qui potrebbero produrre un numero significativamente ridotto di mutazioni farmaco-resistenti di fuga rispetto al contemporaneo antiretrovirali clinicamente valutato.

Una regione simmetrica dell&#39;interfaccia dimerizzazione integrasi dell&#39;HIV-1 &egrave; indispensabile per la replicazione virale.

Hiv-1 &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30E9;&#x30FC;&#x30BC; &#xFF08;IN&#xFF09; &#x306F;&#x3001;&#x73FE;&#x4EE3;&#x7684;&#x306A;&#x6297;&#x30EC;&#x30C8;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x85AC;&#x5264;&#x30C7;&#x30B6;&#x30A4;&#x30F3;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x76EE;&#x6A19;&#x3067;&#x3059;&#x3002;&#x6B74;&#x53F2;&#x7684;&#x306B;&#x3001;&#x9175;&#x7D20;&#x3092;&#x4E0D;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x52AA;&#x529B;&#x306F;&#x6D3B;&#x6027;&#x90E8;&#x4F4D;&#x3092;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x306B;&#x7126;&#x70B9;&#x3092;&#x5F53;&#x3066;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x81E8;&#x5E8A;&#x7684;&#x306B;&#x85AC;&#x3067;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x30A2;&#x30ED;&#x30B9;&#x30C6;&#x30EA;&#x30C3;&#x30AF;&#x963B;&#x5BB3;&#x5264;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30AF;&#x30E9;&#x30B9;&#x304C;&#x6297;&#x30EC;&#x30C8;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x5206;&#x91CE;&#x306B;&#x73FE;&#x4EE3;&#x7684;&#x306A;&#x8010;&#x6027;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x7CFB;&#x7D71;&#x306E;&#x51FA;&#x73FE;&#x306B;&#x7167;&#x3089;&#x3057;&#x3066;&#x4E8B;&#x524D;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x306A;&#x3063;&#x305F;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x6B8B;&#x57FA;&#x3001;&#x9175;&#x7D20;&#x3068;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x8907;&#x88FD;&#x5168;&#x4F53;&#x3068;&#x3057;&#x3066;&#x306E;&#x7FA9;&#x52D9;&#x306E;&#x591A;&#x91CF;&#x4F53;&#x5F62;&#x6210;&#x306B;&#x3068;&#x3063;&#x3066;&#x91CD;&#x8981;&#x3068;&#x3057;&#x3066;&#x3001;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6; &#x30B5;&#x30A4;&#x30C8;&#x304B;&#x3089;&#x9060;&#x304F;&#x3067;&#x306E;&#x7DCA;&#x5BC6;&#x306A;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x306E;&#x91CD;&#x8981;&#x6027;&#x3092;&#x7279;&#x5FB4;&#x304C;&#x3042;&#x308B;.&#x5177;&#x4F53;&#x7684;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F; 6 &#x6B8B;&#x57FA;&#x306E; 2 &#x3064;&#x306E;&#x5869;&#x6A4B;&#x306B;&#x3088;&#x3063;&#x3066;&#x4E26;&#x3076; 4 &#x5C64;&#x306E;&#x82B3;&#x9999;&#x65CF;&#x76F8;&#x4E92;&#x306E;&#x4F5C;&#x66F2;&#x306B;&#x304A;&#x3051;&#x308B;&#x4E8C;&#x91CF;&#x4F53;&#x5316;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x306F;&#x3001;&#x975E;&#x5E38;&#x306B;&#x5BFE;&#x79F0;&#x9818;&#x57DF;&#x5185;&#x306E;&#x69CB;&#x6210;&#x304C;&#x5927;&#x5E45;&#x306B;&#x9069;&#x5207;&#x306A; HIV-1 &#x306E;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x5BC4;&#x4E0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x79C1;&#x9054;&#x306E;&#x30AA;&#x30EA;&#x30B4;&#x30E1;&#x30EA;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x30C1;&#x30A7;&#x30C3;&#x30AF;&#x3057;&#x3001;&#x3053;&#x306E;&#x9818;&#x57DF;&#x306B;&#x6CBF;&#x3063;&#x3066;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x7F6E;&#x63DB;&#x306E;&#x975E;&#x5E38;&#x306B;&#x4F4E;&#x3044;&#x8A31;&#x5BB9;&#x5DEE;&#x304C;&#x3042;&#x308B;&#x3068;&#x5224;&#x65AD;&#x3057;&#x305F;&#x5B9A;&#x91CF;&#x7684;&#x767A;&#x5149;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x5229;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x4FDD;&#x5B88;&#x7684;&#x306A;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x6B8B;&#x57FA;&#x7F6E;&#x63DB;&#x306E;&#x591A;&#x91CF;&#x4F53;&#x5F62;&#x6210;&#x3001;&#x975E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x9175;&#x7D20;&#x3068;&#x975E;&#x8907;&#x88FD;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x7D50;&#x679C;&#x60AA;&#x5F71;&#x97FF;&#x3002;&#x6211;&#x3005; &#x306F;&#x3053;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;, &#x7279;&#x5FB4;&#x3068;&#x5DE6;&#x53F3;&#x5BFE;&#x79F0;&#x306E;&#x4E8C;&#x91CF;&#x4F53;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30FC;&#x30B9;&#x9818;&#x57DF;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x5909;&#x7570;&#x306E;&#x975E;&#x5E38;&#x306B;&#x4F4E;&#x3044;&#x516C;&#x5DEE;&#x3068;&#x3053;&#x3053;&#x3067;&#x306E;&#x8A73;&#x7D30;&#x306A;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x30CD;&#x30C3;&#x30C8;&#x30EF;&#x30FC;&#x30AF;&#x3092;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306B;&#x8A2D;&#x8A08;&#x3055;&#x308C;&#x85AC;&#x3092;&#x671F;&#x5F85;&#x3067;&#x304D;&#x308B;&#x85AC;&#x5264;&#x8010;&#x6027;&#x30A8;&#x30B9;&#x30B1;&#x30FC;&#x30D7;&#x5909;&#x7570;&#x306E;&#x73FE;&#x4EE3;&#x7684;&#x306A;&#x81E8;&#x5E8A;&#x8A55;&#x4FA1;&#x6297;&#x30EC;&#x30C8;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x85AC;&#x306B;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x6709;&#x610F;&#x306A;&#x6E1B;&#x5C11;&#x3092;&#x3082;&#x305F;&#x3089;&#x3059;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

1 HIV &#x30A4;&#x30F3;&#x30C6;&#x30B0;&#x30E9;&#x30FC;&#x30BC; &#x30C0;&#x30A4;&#x30DE;&#x30FC;&#x5316;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x306E;&#x5BFE;&#x79F0;&#x9818;&#x57DF;&#x306F;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x8907;&#x88FD;&#x306B;&#x4E0D;&#x53EF;&#x6B20;&#x3067;&#x3059;&#x3002;

1 HIV integrase (IN) &#xD604;&#xB300; &#xD22C;&#xC5EC; &#xC57D;&#xBB3C; &#xB514;&#xC790;&#xC778; &#xC5F0;&#xAD6C;&#xB97C; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xB300;&#xC0C1; &#xC774;&#xB2E4;. &#xC5ED;&#xC0AC;&#xC801;&#xC73C;&#xB85C;, &#xB178;&#xB825;&#xC744; &#xBE44;&#xD65C;&#xC131;&#xD654;&#xB294; &#xD6A8;&#xC18C;&#xC758; &#xD65C;&#xC131; &#xC0AC;&#xC774;&#xD2B8; &#xCC28;&#xB2E8; &#xC2DC; &#xC9D1;&#xC911;. &#xADF8;&#xB7EC;&#xB098;, &#xADF8;&#xAC83;&#xC740; &#xBA85;&#xBC31;&#xD55C; &#xADF8; allosteric &#xC5B5;&#xC81C;&#xC81C;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xD074;&#xB798;&#xC2A4; &#xD544;&#xC694;&#xAC00; &#xC788;&#xC744; &#xAC83;&#xC785;&#xB2C8;&#xB2E4; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBCC0;&#xC885; &#xD604;&#xB300; &#xB0B4;&#xC131;&#xC758; &#xCD9C;&#xD604;&#xC5D0; &#xBE44;&#xCD94;&#xC5B4; &#xB808;&#xD2B8; &#xD544;&#xB4DC; &#xC0AC;&#xC804; &#xC784;&#xC0C1; &#xC758;&#xC57D;&#xD488;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xB294; &#xB418;&#xACE0;&#xC788;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC;&#xAC00; &#xC794;&#xB958;&#xBB3C;, &#xD6A8;&#xC18C; &#xBC0F; &#xC804;&#xCCB4; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBCF5;&#xC81C;&#xC758; &#xC758;&#xBB34; multimerization&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xD65C;&#xC131; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0;&#xC11C; &#xBA40;&#xB9AC; &#xB5A8;&#xC5B4;&#xC838; &#xC788;&#xB294; &#xAC00;&#xAE4C;&#xC6B4; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xC758; &#xC911;&#xC694;&#xC131;&#xC774; &#xD2B9;&#xC9D5;&#xC785;&#xB2C8;&#xB2E4;. &#xD2B9;&#xD788;, &#xC6B0;&#xB9AC; &#xACB0;&#xC815; &#xB450; &#xC18C;&#xAE08; &#xAD50;&#xB7C9;&#xC5D0; &#xC758;&#xD574; &#xD615;&#xBC8C; 4 &#xACC4;&#xCE35; &#xC544;&#xB85C;&#xB9C8; &#xC791;&#xC6A9;&#xC73C;&#xB85C; &#xC774;&#xB8E8;&#xC5B4;&#xC9C4; IN dimerization &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xC5D0; &#xB9E4;&#xC6B0; &#xB300;&#xCE6D; &#xC601;&#xC5ED; &#xB0B4;&#xC5D0;&#xC11C; 6 &#xC794;&#xB958;&#xBB3C;&#xC758; &#xAD6C;&#xC131;&#xC744; &#xD06C;&#xAC8C; &#xC801;&#xC808; &#xD55C; HIV-1 &#xBCF5;&#xC81C;&#xC5D0; &#xAE30;&#xC5EC; &#xD55C;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC; oligomerization&#xC5D0;&#xC11C; &#xAC80;&#xC0AC; &#xD558;&#xB294; &#xC544;&#xBBF8;&#xB178;&#xB97C; &#xD568;&#xC720;&#xD55C; &#xC0B0; &#xC131; &#xB300;&#xCCB4;&#xB97C; &#xB530;&#xB77C;&#xC774; &#xC9C0;&#xC5ED;&#xC5D0; &#xB300; &#xD55C; &#xB9E4;&#xC6B0; &#xB0AE;&#xC740; &#xD5C8;&#xC6A9; &#xC624;&#xCC28; &#xACB0;&#xC815; &#xC591;&#xC801; &#xBC1C;&#xAD11; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xD65C;&#xC6A9;. &#xC2EC;&#xC9C0;&#xC5B4; &#xBCF4;&#xC218;&#xC801;&#xC778; &#xC794;&#xB958;&#xBB3C; &#xB300;&#xCCB4; multimerization, &#xBE44;&#xD65C;&#xC131; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xD6A8;&#xC18C; &#xBC0F; &#xBE44; &#xC77C;&#xCC28; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xACB0;&#xACFC;&#xC5D0; &#xBD80;&#xC815;&#xC801;&#xC778; &#xC601;&#xD5A5;&#xC744;. &#xC6B0;&#xB9AC;&#xB294;&#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &#xD2B9;&#xC9D5; &#xB300;&#xCE6D; dimeric &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4; &#xC601;&#xC5ED;&#xC5D0;&#xC11C; &#xC544;&#xBBF8;&#xB178;&#xC0B0; &#xBCC0;&#xC774; &#xB300; &#xD55C; &#xB9E4;&#xC6B0; &#xB0AE;&#xC740; &#xB0B4;&#xC131; &#xACE0; &#xB530;&#xB77C;&#xC11C; &#xC5EC;&#xAE30;&#xC5D0;&#xC11C; &#xC124;&#xBA85; &#xD558;&#xB294; &#xC544;&#xBBF8;&#xB178;&#xB97C; &#xD568;&#xC720;&#xD55C; &#xC0B0; &#xC131; &#xB124;&#xD2B8;&#xC6CC;&#xD06C;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xC124;&#xACC4; &#xB41C; &#xC57D;&#xBB3C; &#xC57D;&#xBB3C; &#xB0B4;&#xC131; &#xD0C8;&#xCD9C; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xD604;&#xB300; &#xC784;&#xC0C1; &#xD3C9;&#xAC00; antiretrovirals&#xC5D0; &#xBE44;&#xD574; &#xD06C;&#xAC8C; &#xAC10;&#xC18C; &#xB41C; &#xC218;&#xB97C; &#xC608;&#xC0C1; &#xC218; &#xB098;&#xD0C0;&#xB0AC;&#xC2B5;&#xB2C8;&#xB2E4;.

1 HIV Integrase Dimerization &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xC758; &#xB300;&#xCE6D; &#xC601;&#xC5ED; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBCF5;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC785;&#xB2C8;&#xB2E4;.

Integrasa del VIH-1 (IN) es un objetivo importante para la investigaci&oacute;n de dise&ntilde;o de drogas antirretrovirales contempor&aacute;neo. Hist&oacute;ricamente, los esfuerzos inactivar la enzima han centrado a bloquear su sitio activo. Sin embargo, se ha puesto de manifiesto que nuevas clases de inhibidores alost&eacute;ricos ser&aacute; necesarias avanzar en el campo de antirretrovirales a la luz de la aparici&oacute;n de cepas virales resistentes a contempor&aacute;neo cl&iacute;nico utilizado en medicamentos. En este estudio hemos caracterizado la importancia de una estrecha red de en residuos, distantes del sitio activo, tan importante para la multimerization obligatoria de la enzima y la replicaci&oacute;n viral en su conjunto. En concreto, hemos determinado que la configuraci&oacute;n de seis residuos dentro de una regi&oacute;n altamente sim&eacute;trica en la interfaz de dimerizaci&oacute;n IN, compuesta por una interacci&oacute;n arom&aacute;tica cuatro niveles, flanqueada por dos puentes de sal, contribuye significativamente a la adecuada replicaci&oacute;n del VIH-1. Adem&aacute;s, hemos utilizado un an&aacute;lisis cuantitativo de la luminiscencia para examinar la Oligomerizaci&oacute;n y han determinado que existe una tolerancia muy baja para sustituciones de amino&aacute;cidos a lo largo de esta regi&oacute;n. Incluso los conservadores residuo sustituciones impactado negativamente en multimerization, dando por resultado una enzima viral inactiva y un virus no replicativo. Hemos demostrado que hay una muy baja tolerancia a la variaci&oacute;n del amino&aacute;cido en la regi&oacute;n de interfaz dim&eacute;rico sim&eacute;trica caracterizada en este estudio, y por lo tanto medicamentos dise&ntilde;ados para atacar la red del amino&aacute;cido detallada aqu&iacute; podr&iacute;an esperarse un n&uacute;mero significativamente menor de mutaciones de escape resistente a los medicamentos antirretrovirales contempor&aacute;neo evaluado cl&iacute;nicamente en comparaci&oacute;n con la producci&oacute;n.

Una regi&oacute;n sim&eacute;trica de la interfaz de dimerizaci&oacute;n de la integrasa del VIH-1 es esencial para la replicaci&oacute;n Viral.

On the basis of an initial molecular modeling study suggesting the favorable binding of the "privileged" fragment 8-hydroxyquinoline with HIV-1 integrase (IN) at the IN-lens epithelium-derived growth factor/p75 (LEDGF/p75) interface , we developed a set of modified 8-hydroxyquinoline fragments demonstrating micromolar IC50 values for inhibition of the IN-LEDGF/p75 interaction, but significant cytotoxicity was associated with these initial compounds. Diverse modifications at the C5 and C7 carbons of the 8-hydroxyquinoline core improved potency, but reduction of diversity to only modifications at the C5 position ultimately yielded potent inhibitors with low cytotoxicity. Two of these particular compounds, 5-((p-tolylamino)methyl)quinolin-8-ol and 5-(((3,4-dimethylphenyl)amino)methyl)quinolin-8-ol, inhibited viral replication in MT-4 cells with low micromolar EC50. This is the first study providing evidence for 8-hydroxyquinolines as novel inhibitors of the IN-LEDGF/p75 interaction. Our lead compounds are druglike, have low molecular weights, and are amenable to various substitutions suitable for enhancing their potency and selectivity.

Fragment-based discovery of 8-hydroxyquinoline inhibitors of the HIV-1 integrase-lens epithelium-derived growth factor/p75 (IN-LEDGF/p75) interaction.

Though much progress has been made in the inhibition of HIV-1 integrase catalysis, clinical resistance mutations have limited the promise of long-term drug prescription. Consequently, allosteric inhibition of integrase activity has emerged as a promising approach to antiretroviral discovery and development. Specifically, inhibitors of the interaction between HIV-1 integrase and cellular cofactor LEDGF/p75 have been validated to diminish proviral integration in cells and deliver a potent reduction in viral replicative capacity. Here, we have contributed to the development of novel allosteric integrase inhibitors with a high-throughput AlphaScreen-based random screening approach, with which we have identified novel 5-carbonyl-1H-imidazole-4-carboxamides capable of inhibiting the HIV-1 integrase-LEDGF/p75 interaction in vitro. Following a structure-activity relationship analysis of the initial 1H-imidazole-4,5-dicarbonyl core, we optimized the compound's structure through an industrial database search, and we went further to synthesize a selective and non-cytotoxic panel of inhibitors with enhanced potency.

Discovery of a novel 5-carbonyl-1H-imidazole-4-carboxamide class of inhibitors of the HIV-1 integrase-LEDGF/p75 interaction.

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3' processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.

Biochemical characterization of novel retroviral integrase proteins.

Multiple human proteins have been shown to both support and restrict viral replication, and confirmation of virus-associated changes in the expression of these genes is relevant for future therapeutic efforts. In this study a well-characterized panel of 49 individuals either infected with HIV-1 or uninfected was compiled and analyzed for the effect of HIV infection status, viral load, and antiretroviral treatment on specific gene expression. mRNA was extracted and reverse transcribed from purified CD4+ cells, and quantitative real-time PCR was utilized to scrutinize differences in the expression of four host genes that have been demonstrated to either stimulate (HSP90 and LEDGF/p75) or restrict (p21/WAF1 and APOBEC3G) proviral integration. HIV infection status was associated with slight to moderate alterations in the expression of all four genes. After adjusting for age, mRNA expression levels of HSP90, LEDGF/p75 and APOBEC3G were found to all be decreased in infected patients compared to healthy controls by 1.43-, 1.26-, and 4.71-fold, respectively, while p21/WAF1 expression was increased 2.35-fold. Furthermore, individuals receiving raltegravir exhibited a 1.28-fold reduction in LEDGF/p75 compared to those on non-raltegravir antiretroviral treatment. Identification of these and similar HIV-induced changes in gene expression may be valuable for delineating the extent of host cell molecular mechanisms stimulating viral replication.

Alteration of select gene expression patterns in individuals infected with HIV-1.

Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions -3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration.

Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding.

HIV integrase (IN) is an essential enzyme for the viral replication. Currently, three IN inhibitors have been approved for treating HIV-1 infection. All three drugs selectively inhibit the strand transfer reaction by chelating a divalent metal ion in the enzyme active site. Flavonoids are a well-known class of natural products endowed with versatile biological activities. Their β-ketoenol or catechol structures can serve as a metal chelation motif and be exploited for the design of novel IN inhibitors. Using the metal chelation as a common pharmacophore, we introduced appropriate hydrophobic moieties into the flavonol core to design natural product-based novel IN inhibitors. We developed selective and efficient syntheses to generate a series of mono 3/5/7/3'/4'-substituted flavonoid derivatives. Most of these new compounds showed excellent HIV-1 IN inhibitory activity in enzyme-based assays and protected against HIV-1 infection in cell-based assays. The 7-morpholino substituted 7c showed effective antiviral activity (EC50=0.826 μg/mL) and high therapeutic index (TI>242). More significantly, these hydroxyflavones block the IN-LEDGF/p75 interaction with low- to sub-micromolar IC50 values and represent a novel scaffold to design new generation of drugs simultaneously targeting the catalytic site as well as protein-protein interaction domains.

Design and discovery of flavonoid-based HIV-1 integrase inhibitors targeting both the active site and the interaction with LEDGF/p75.

Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic.

Molecular mechanisms of retroviral integration site selection.

Interferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection.

Host and viral determinants for MxB restriction of HIV-1 infection.

Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joining and generates a short (4-6 bp) duplication of host DNA flanking the provirus. We previously determined two key parameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiency virus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integration sites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase. Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings with respect to length of target site duplication (TSD).

Key determinants of target DNA recognition by retroviral intasomes.

Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

Structural basis for retroviral integration into nucleosomes.

One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency.

Sites of retroviral DNA integration: From basic research to clinical applications.

Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.

The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency.

Article	Year
Raltegravir, elvitegravir, and metoogravir: the birth of "me-too" HIV-1 integrase inhibitors. Retrovirology\| PubMed ID: 19265512	2009
HIV-1 integrase inhibitors: 2007-2008 update. Medicinal research reviews\| PubMed ID: 20135632	2010
A Symmetric Region of the HIV-1 Integrase Dimerization Interface Is Essential for Viral Replication. PloS one\| PubMed ID: 23028829	2012
Fragment-based discovery of 8-hydroxyquinoline inhibitors of the HIV-1 integrase-lens epithelium-derived growth factor/p75 (IN-LEDGF/p75) interaction. Journal of medicinal chemistry\| PubMed ID: 23445471	2013
Discovery of a novel 5-carbonyl-1H-imidazole-4-carboxamide class of inhibitors of the HIV-1 integrase-LEDGF/p75 interaction. Bioorganic & medicinal chemistry\| PubMed ID: 23985689	2013
Biochemical characterization of novel retroviral integrase proteins. PloS one\| PubMed ID: 24124581	2013
Alteration of select gene expression patterns in individuals infected with HIV-1. Journal of medical virology\| PubMed ID: 24482297	2014
Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding. Nucleic acids research\| PubMed ID: 24520116	2014
Design and discovery of flavonoid-based HIV-1 integrase inhibitors targeting both the active site and the interaction with LEDGF/p75. Bioorganic & medicinal chemistry\| PubMed ID: 24794743	2014
Molecular mechanisms of retroviral integration site selection. Nucleic acids research\| PubMed ID: 25147212	2014
Host and viral determinants for MxB restriction of HIV-1 infection. Retrovirology\| PubMed ID: 25348155	2014
Key determinants of target DNA recognition by retroviral intasomes. Retrovirology\| PubMed ID: 25924943	2015
Structural basis for retroviral integration into nucleosomes. Nature\| PubMed ID: 26061770	2015
Sites of retroviral DNA integration: From basic research to clinical applications. Critical reviews in biochemistry and molecular biology\| PubMed ID: 26508664	2016
A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 26858452	2016
The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS chemical biology\| PubMed ID: 26910179	2016

Erik Serrao

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