© À. Í. Ãîðøêîâ À. Â. Ïåòðîâà À. Â....

ÐÍÊ-ÈÍÒÅÐÔÅÐÅÍÖÈß È ÏÀÒÎÃÅÍÅÇ ÂÈÐÓÑÀ ÃÐÈÏÏÀ À

© À. Í. Ãîðøêîâ,1, 2, * À. Â. Ïåòðîâà,1 À. Â. Âàñèí1, 3

1 Íàó÷íî-èññëåäîâàòåëüñêèé èíñòèòóò ãðèïïà Ìèíèñòåðñòâà çäðàâîîõðàíåíèÿ ÐÔ,Ñàíêò-Ïåòåðáóðã, 197376,

2 Èíñòèòóò öèòîëîãèè ÐÀÍ, Ñàíêò-Ïåòåðáóðã, 194064, è3 Êàôåäðà ìîëåêóëÿðíîé áèîëîãèè Ñ.-Ïåòåðáóðãñêîãî ïîëèòåõíè÷åñêîãî óíèâåðñèòåòà Ïåòðà Âåëèêîãî,

Ñàíêò-Ïåòåðáóðã, 195251;* ýëåêòðîííûé àäðåñ: [email protected]

À. Í. Ãîðøêîâ è äð.ÐÍÊ-èíòåðôåðåíöèÿ è ïàòîãåíåç âèðóñà ãðèïïà À

ßâëåíèå ÐÍÊ-èíòåðôåðåíöèè áûëî îòêðûòî â 90-å ãîäû XX â. â èññëåäîâàíèÿõ íà íåìàòîäå Caenor-habditis elegans. Òåðìèíîëîãè÷åñêè, èçíà÷àëüíî ÐÍÊ-èíòåðôåðåíöèÿ áûëà îïðåäåëåíà êàê ñåëåêòèâíàÿäåãðàäàöèÿ ìÐÍÊ-òðàíñêðèïòà ïðè ïîÿâëåíèè â êëåòêàõ êîðîòêîé èíòåðôåðèðóþùåé ÐÍÊ (êèÐÍÊ) —êîðîòêîé (20—25 ï. í.) ýêçîãåííîé äâóõíèòåâîé ÐÍÊ, êîìïëåìåíòàðíîé ê åãî ýêçîííûì ó÷àñòêàì. Çàïðîøåäøèå ñ òîãî âðåìåíè 2 äåñÿòèëåòèÿ ïîíÿòèå ÐÍÊ-èíòåðôåðåíöèè ïðèîáðåëî çíà÷èòåëüíî áîëååøèðîêîå çíà÷åíèå; îíà áûëà îñìûñëåíà â êà÷åñòâå îäíîãî èç âàæíåéøèõ è óíèâåðñàëüíûõ çâåíüåâïîñòòðàíñêðèïöèîííîé ðåãóëÿöèè àêòèâíîñòè ãåíîâ, øèðîêî ðàñïðîñòðàíåííîé ñðåäè âñåõ ãðóïï ýóêà-ðèîò, à òàêæå âèðóñîâ. Íàñòîÿùèé îáçîð âêëþ÷àåò â ñåáÿ êðàòêóþ õàðàêòåðèñòèêó ðàçëè÷íûõ ìåõàíèç-ìîâ àíòèâèðóñíîé è ïðîâèðóñíîé ÐÍÊ-èíòåðôåðåíöèè; äàëåå ìû ðàññìàòðèâàåì çíà÷åíèå ÐÍÊ-èíòåð-ôåðåíöèè â áèîëîãèè âèðóñà ãðèïïà À, à òàêæå âîçìîæíîñòè è òðóäíîñòè èñïîëüçîâàíèÿ ÐÍÊ-èíòåðôå-ðåíöèè êàê íîâîé òåðàïåâòè÷åñêîé ñòðàòåãèè â áîðüáå ñ ãðèïïîì.

Ê ë þ ÷ å â û å ñ ë î â à: ÐÍÊ-èíòåðôåðåíöèÿ, âèðóñ ãðèïïà À, ìèêðîÐÍÊ, êèÐÍÊ, èíòåðôåðîíîâûéîòâåò.

Ï ð è í ÿ ò û å ñ î ê ð à ù å í è ÿ: ÂÃÀ — âèðóñ ãðèïïà À, ÂÃÑ — âèðóñ ãåïàòèòà Ñ, ÂÈ× — âèðóñ èì-ìóíîäåôèöèòà ÷åëîâåêà, ÎÐÑ — îòêðûòàÿ ðàìêà ñ÷èòûâàíèÿ, ï. í. — ïàðû íóêëåîòèäîâ, ÏÒÃÑ — ïîñò-òðàíñêðèïöèîííûé ãåííûé ñàéëåíñèíã, äíÐÍÊ — äâóõíèòåâàÿ ÐÍÊ, êèÐÍÊ — êîðîòêàÿ èíòåðôåðèðó-þùàÿ ÐÍÊ, êøÐÍÊ — êîðîòêàÿ øïèëå÷íàÿ ÐÍÊ, ìÐÍÊ — ìàòðè÷íàÿ ÐÍÊ, ÝÑÊ — ýìáðèîíàëüíûåñòâîëîâûå êëåòêè, piÐÍÊ — Piwi-âçàèìîäåéñòâóþùàÿ ÐÍÊ, pri-miRNA — ïåðâè÷íûé ìèêðîÐÍÊ-òðàíñ-êðèïò, pre-miRNA — ïðåäøåñòâåííèê ìèêðîÐÍÊ, RdRp — ÐÍÊ-çàâèñèìàÿ ÐÍÊ-ïîëèìåðàçà, RISC —ÐÍÊ-èíäóöèðîâàííûé êîìïëåêñ ñàéëåíñèíãà, UTR — íåòðàíñëèðóåìàÿ îáëàñòü, MRE — ìèêðîÐÍÊ-óç-íàþùèé ýëåìåíò.

Âèðóñ ãðèïïà À (ÂÃÀ), ðåãóëÿðíî âûçûâàþùèé ýïè-äåìèè è ïàíäåìèè, ÿâëÿåòñÿ ñåðüåçíîé ãëîáàëüíîé ìåäè-öèíñêîé ïðîáëåìîé. Ïðè òÿæåëîì òå÷åíèè áîëåçíè ÂÃÀâûçûâàåò íå òîëüêî èíôåêöèþ ðåñïèðàòîðíîãî òðàêòà, íîè ñèñòåìíûå íàðóøåíèÿ, ñïîñîáíûå ñòàòü ïðè÷èíîéñìåðòè.

Èñïîëüçóåìûå ñïåöèôè÷åñêèå àíòè-ÂÃÀ ëåêàðñòâåí-íûå ñðåäñòâà íàïðàâëåíû íà áëîêèðîâàíèå ôóíêöèè äâóõâèðóñíûõ áåëêîâ — íåéðàìèíèäàçû (ÍÀ) è èîííîãî êà-íàëà M2. Â ïåðâîì ñëó÷àå èñïîëüçóþò îñåëüòàìèâèð èçàíàìèâèð, âî âòîðîì — ðåìàíòàäèí è àìàíòàäèí. Îäíà-êî ê íàñòîÿùåìó âðåìåíè âîçíèêëè øòàììû ÂÃÀ, óñòîé÷è-âûå ê ýòèì ãðóïïàì ëåêàðñòâ (Saladino et al., 2010; Jacobet al., 2016). Ïîÿâëåíèå òàêèõ øòàììîâ ñòèìóëèðóåò ïîèñêíîâûõ ñòðàòåãèé è ëåêàðñòâ, âëèÿþùèõ íå íà èíãèáèðîâà-íèå âèðóñíûõ áåëêîâ, à íà ïðèíöèïèàëüíî äðóãèå ìèøåíè.

Îòêðûòèå áèîëîãè÷åñêîãî ôåíîìåíà ÐÍÊ-èíòåðôå-ðåíöèè (íàçûâàåìîãî òàêæå ïîñòòðàíñêðèïöèîííûì ãåí-íûì ñàéëåíñèíãîì — ÏÒÃÑ) îáíàðóæèëî íåèçâåñòíûåðàíåå ñëîæíîñòü è ãëóáèíó âçàèìîäåéñòâèé âèðóñîâ èèíôèöèðîâàííûõ èìè êëåòîê, âîâëåêàþùèõ ìèêðîÐÍÊ

êëåòîê-õîçÿåâ, ìèêðîÐÍÊ âèðóñîâ, à òàêæå (â íåêîòîðûõãðóïïàõ îðãàíèçìîâ) âèðóñíûå êîðîòêèå èíòåðôåðèðóþ-ùèå ÐÍÊ (êèÐÍÊ). Èäåè î ïåðñïåêòèâàõ òåðàïåâòè÷åñêî-ãî èñïîëüçîâàíèÿ ÐÍÊ-èíòåðôåðåíöèè äëÿ ïîäàâëåíèÿýêñïðåññèè «íåæåëàòåëüíûõ» ãåíîâ (ìóòàíòíûõ ãåíîâ,îíêîãåíîâ è âèðóñíûõ ãåíîâ) âîçíèêëè âñêîðå ïîñëå ååîòêðûòèÿ (Shuey et al., 2002; Coburn, Cullen, 2003). Ïîòåí-öèàë ÐÍÊ-èíòåðôåðåíöèè â áîðüáå ñ ÂÃÀ àêòèâíî èññëå-äóåòñÿ â ïîñëåäíåå äåñÿòèëåòèå. Ïîìèìî ñàéëåíñèíãàãåíîâ ÂÃÀ ýêñïåðèìåíòàëüíî ðàçðàáàòûâàåòñÿ è ðÿä äðó-ãèõ àíòèâèðóñíûõ ïðèìåíåíèé ÐÍÊ-èíòåðôåðåíöèè —êèÐÍÊ-ñêðèíèíã íîâûõ êëåòî÷íûõ ìèøåíåé äëÿ òåðàïèèãðèïïà, ñîçäàíèå àòòåíóèðîâàííûõ âàêöèí íîâîãî ïîêî-ëåíèÿ è òåðàïåâòè÷åñêàÿ êîìïåíñàöèÿ äåðåãóëÿöèè êëå-òî÷íûõ ìèêðîÐÍÊ ïðè èíôåêöèè ÂÃÀ.

Öåëü íàñòîÿùåãî îáçîðà — îïèñàíèå îñíîâíûõ ìåõà-íèçìîâ ïðîâèðóñíîé è àíòèâèðóñíîé ÐÍÊ-èíòåðôåðåí-öèè, à òàêæå àíàëèç íåäàâíèõ ïóáëèêàöèé, îòðàæàþùèõóñïåõè è òðóäíîñòè èñïîëüçîâàíèÿ ÐÍÊ-èíòåðôåðåíöèèêàê íîâîãî òåðàïåâòè÷åñêîãî ïîäõîäà â áîðüáå ñ âèðóñîìãðèïïà.

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Æèçíåííûé öèêë âèðóñà ãðèïïà À

ÂÃÀ ÿâëÿåòñÿ îáîëî÷å÷íûì âèðóñîì èç ñåìåéñòâà Or-thomyxoviridae. ÂÃÀ èìååò ñåãìåíòèðîâàííûé ãåíîì, ñî-ñòîÿùèé èç 8 ñåãìåíòîâ ÐÍÊ ñ îòðèöàòåëüíîé ïîëÿðíî-ñòüþ. Ãåíîì ÂÃÀ êîäèðóåò ñëåäóþùèå âèðóñíûå áåëêè:ïîâåðõíîñòíûå ãëèêîïðîòåèíû ãåìàããëþòèíèí è íåéðà-ìèíèäàçó, èîííûé êàíàë M2, ìàòðèêñíûé áåëîê M1, íóê-ëåîïðîòåèí NP, ïîëèìåðàçíûé êèñëûé áåëîê PA, ïîëèìå-ðàçíûå îñíîâíûå áåëêè 1 è 2 (PB1 è PB2 ñîîòâåòñòâåííî),áåëîê PB1-F2 (õàðàêòåðíûé íå äëÿ âñåõ øòàììîâ ÂÃÀ),íåñòðóêòóðíûé áåëîê 1 (NS1) è 2 (NS2, íàçûâàåìûé òàê-æå áåëêîì ÿäåðíîãî ýêñïîðòà, NEP).

Æèçíåííûé öèêë ÂÃÀ â èíôèöèðîâàííûõ êëåòêàõèññëåäîâàëè íåîäíîêðàòíî (ñì. îáçîðû: Samji, 2009; Êè-ñåëåâ, 2011; Ùåëêàíîâ è äð., 2015). Êðàòêî, íà÷àëüíàÿòî÷êà öèêëà — àäñîðáöèÿ âèðóñà — îïîñðåäóåòñÿ âçàè-ìîäåéñòâèåì ñóáúåäèíèöû HA1 ãåìàããëþòèíèíà ñ îñòàò-êàìè N-àöåòèëíåéðàìèíîâîé (ñèàëîâîé) êèñëîòû, ëîêà-ëèçîâàííûìè íà ïîâåðõíîñòè êëåòîê-ìèøåíåé. Îñîáåí-íîñòè ýòîãî âçàèìîäåéñòâèÿ, ïðåæäå âñåãî ñâÿçûâàíèåHA1 ñ a2—3- èëè a2—6-ñèàëîçèäàìè, ïðåèìóùåñòâåííîîïðåäåëÿþò âèäîñïåöèôè÷íîñòü, òðîïèçì è â êîíå÷íîìèòîãå — ýïèäåìè÷åñêèé ïîòåíöèàë ðàçëè÷íûõ øòàììîâÂÃÀ.

Ïðîíèêíîâåíèå âèðóñà â êëåòêó ñâÿçàíî ñ ïðîöåññîìýíäîöèòîçà. Èçâåñòíî, ÷òî ýíäîöèòîç ÂÃÀ ìîæåò áûòüêàê êëàññè÷åñêèì êëàòðèíçàâèñèìûì, òàê è êëàòðèííåçà-âèñèìûì (ñ ó÷àñòèåì êàâåîë ëèáî ïîñðåäñòâîì ìàêðîïè-íîöèòîçà). Ýíäîöèòèðîâàííûå âèðèîíû ëîêàëèçóþòñÿ âýíäîñîìàõ, êîòîðûå òðàíñëîöèðóþòñÿ èç ñóáìåìáðàííîéâ ïåðèíóêëåàðíóþ îáëàñòü öèòîïëàçìû. Êëþ÷åâûì ñîáû-òèåì, íåîáõîäèìûì äëÿ âûõîäà âèðóñíûõ íóêëåîêàïñè-äîâ èç ýíäîñîì, ÿâëÿåòñÿ êîíôîðìàöèîííûé ïåðåõîäñóáúåäèíèöû HA2 ãåìàããëþòèíèíà â óñëîâèÿõ íèçêîãîpH ïîçäíåé ýíäîñîìû, ïðèâîäÿùèé ê ýêñïîíèðîâàíèþðàíåå ñêðûòîãî ïåïòèäà ñëèÿíèÿ è âñòðàèâàíèþ äàííîãîïåïòèäà â ìåìáðàíó ýíäîñîìû, ò. å. ê ñëèÿíèþ ìåìáðàíâèðèîíà è ýíäîñîìû. Îäíîâðåìåííî íèçêèé pH ýíäîñîìûîòêðûâàåò âèðóñíûé ïðîòîííûé êàíàë M2, ÷òî ïðèâîäèòê ïîíèæåíèþ pH âíóòðè âèðèîíà è îñâîáîæäåíèþ âèðóñ-íûõ ðèáîíóêëåîïðîòåèíîâ (ÐÍÏ) îò ìàòðèêñíîãî áåëêàM1. Â èòîãå âèðóñíûå ÐÍÏ-êîìïëåêñû âûõîäÿò èç ïðî-ñâåòà ýíäîñîìû â öèòîçîëü è çàòåì òðàíñïîðòèðóþòñÿ âÿäðî. Òðàíñïîðò ÐÍÏ â ÿäðî îïîñðåäóåòñÿ íåñêîëüêèìèêàðèîôåðèíàìè, â ÷àñòíîñòè èìïîðòèíàìè aè b.

Òðàíñêðèïöèÿ âèðóñíûõ ÐÍÊ â ÿäðå îñóùåñòâëÿåòñÿâèðóñíîé ÐÍÊ-çàâèñèìîé ÐÍÊ-ïîëèìåðàçîé (RdRp) —ãåòåðîòðèìåðîì èç PA, PB1 è PB2. Äëÿ èíèöèàöèè òðàíñ-êðèïöèè íåîáõîäèì ïðîöåññ «êýï-ñíýò÷èíãà» («ïîõèùå-íèÿ êýïîâ»), â õîäå êîòîðîãî âèðóñíàÿ PB2 ñâÿçûâàåòñÿ ñêýïèðîâàííûì 5R-êîíöîì êëåòî÷íîé ìÐÍÊ, à PA îòðåçàåòêýï-ñòðóêòóðó è ïåðâûå 10—15 íóêëåîòèäîâ êëåòî÷íîéìÐÍÊ, êîòîðûå èñïîëüçóþòñÿ â êà÷åñòâå ïðàéìåðà äëÿïîñëåäóþùåé òðàíñêðèïöèè âèðóñíîé ìÐÍÊ. Íà 3R-êîíöåâèðóñíàÿ ìÐÍÊ ïîëó÷àåò ïîëèÀ-õâîñò çà ñ÷åò «ïðîáóê-ñîâêè» RdRp íà ïîëèU-ïîñëåäîâàòåëüíîñòè âèðóñíîéÐÍÊ. Äëÿ ýêñïîðòà âèðóñíûõ ìÐÍÊ èç ÿäðà èñïîëüçóåòñÿðÿä êëåòî÷íûõ áåëêîâ, â ÷àñòíîñòè TREX è NXF1/TAP.

Íà áîëåå ïîçäíèõ ñòàäèÿõ ÂÃÀ-èíôåêöèè RdRp ïåðå-êëþ÷àåòñÿ ñ òðàíñêðèïöèîííîé íà ðåïëèêàöèîííóþ àê-òèâíîñòü. Ðåïëèêàöèÿ âèðóñíûõ ÐÍÊ â îòëè÷èå îò òðàíñ-êðèïöèè íå òðåáóåò ïðàéìåðà è ôèíàëüíî ïðèâîäèò êñèíòåçó òî÷íûõ êîïèé âèðóñíîé ÐÍÊ, íå íåñóùèõ5R-êýï-ñòðóêòóðû è 3R-ïîëèÀ-õâîñòà.

Ñèíòåç âèðóñíûõ áåëêîâ ïðîèñõîäèò íà ðèáîñîìàõ âöèòîïëàçìå, ïîñëå ÷åãî áåëêîâûå êîìïîíåíòû ÐÍÏ-êîì-ïëåêñà íàïðàâëÿþòñÿ â ÿäðî äëÿ àññîöèàöèè ñî âíîâüñèíòåçèðîâàííûìè âèðóñíûìè ÐÍÊ, à ïîâåðõíîñòíûåáåëêè âèðóñà òðàíñïîðòèðóþòñÿ ïî ñåêðåòîðíîìó ïóòè,ãëèêîçèëèðóþòñÿ â àïïàðàòå Ãîëüäæè è âñòðàèâàþòñÿ âàïèêàëüíóþ ïëàçìàòè÷åñêóþ ìåìáðàíó. Ñîáðàííûå âÿäðå ÐÍÏ-êîìïëåêñû òàêæå äîñòàâëÿþòñÿ ê ìåñòàì ïî÷-êîâàíèÿ íîâûõ âèðèîíîâ íà àïèêàëüíîé ìåìáðàíå, ïðè-÷åì ñàéòàìè ñáîðêè è ïî÷êîâàíèÿ âèðóñíîãî ïîòîìñòâàÿâëÿþòñÿ ìåìáðàííûå ðàôòû, îáîãàùåííûå õîëåñòåðè-íîì. Âàæíåéøèì ôàêòîðîì â îòïî÷êîâûâàíèè âèðèîíîâîò ìåìáðàíû ÿâëÿåòñÿ ôåðìåíòàòèâíàÿ àêòèâíîñòü íåéðà-ìèíèäàçû, âûçûâàþùàÿ ðàñùåïëåíèå a-êåòîçèäíîé ñâÿçèìåæäó òåðìèíàëüíûì îñòàòêîì ñèàëîâîé êèñëîòû è ñëå-äóþùèì ìîíîñàõàðèäíûì îñòàòêîì. Ñ ïîìîùüþ ìóòàöè-îííîãî àíàëèçà áûëà îáíàðóæåíà òàêæå êëþ÷åâàÿ ðîëüöèòîïëàçìàòè÷åñêîãî õâîñòà áåëêà M2 è ìàòðèêñíîãîáåëêà M1 â ïðîöåññå ïî÷êîâàíèÿ äî÷åðíèõ âèðèîíîâÂÃÀ.

Íà âñåõ ýòàïàõ æèçíåííîãî öèêëà ÂÃÀ âèðóñ òåñíåé-øèì îáðàçîì âçàèìîäåéñòâóåò ñ áîëüøèì êîëè÷åñòâîìýôôåêòîðíûõ è ñèãíàëüíûõ áåëêîâûõ ñèñòåì èíôè-öèðîâàííîé êëåòêè, èñïîëüçóÿ è ïåðåñòðàèâàÿ èõ â öåëÿõñîáñòâåííîãî ðàçìíîæåíèÿ (Watanabe et al., 2010). Â êà-÷åñòâå ïðèìåðîâ òàêèõ ñèñòåì ìîæíî íàçâàòü ìèòîãåíàê-òèâèðóåìûå ñèãíàëüíûå ïóòè (MAPK), ôîñôàòèäèë-èíî-çèòîëüíûé ñèãíàëüíûé ïóòü, ìíîãî÷èñëåííûå ìàëûåÃÒÔàçû è èõ ðåãóëÿòîðû, àêòèíîâûé è òóáóëèíîâûé êîì-ïîíåíòû öèòîñêåëåòà, ñèñòåìû âåçèêóëÿðíîãî òðàíñïîðòàè ñîðòèðîâêè è çíà÷èòåëüíîå êîëè÷åñòâî äðóãèõ áåëêî-âûõ ïëàòôîðì. Â ÷àñòíîñòè, èçâåñòíî ìíîæåñòâî âçàèìî-äåéñòâèé âèðóñíîãî íåñòðóêòóðíîãî áåëêà NS1 ñ ðàçëè÷-íûìè êëåòî÷íûìè áåëêàìè, ïðèâîäÿùèõ ê ïîäàâëåíèþèíòåðôåðîíîâîãî îòâåòà êëåòêè, èíãèáèðîâàíèþ àïîïòî-çà è ê ïîäàâëåíèþ ñïëàéñèíãà è ýêñïîðòà èç ÿäðà êëåòî÷-íûõ ìÐÍÊ.

Çíà÷èòåëüíàÿ ÷àñòü êëåòî÷íûõ ãåíîâ, âîâëå÷åííûõ âðåïëèêàöèþ ÂÃÀ, ïîñòòðàíñêðèïöèîííî êîíòðîëèðóåòñÿìèêðîÐÍÊ. Ðåçêîå èçìåíåíèå ïðîôèëÿ äàííûõ êëåòî÷-íûõ ìèêðîÐÍÊ ÿâëÿåòñÿ âàæíîé ñòîðîíîé ïàòîãåíåçàÂÃÀ. Ñîîòâåòñòâåííî ïåðñïåêòèâû òåðàïåâòè÷åñêîãî èñ-ïîëüçîâàíèÿ ÐÍÊ-èíòåðôåðåíöèè ñâÿçàíû êàê ñ ÏÒÃÑâèðóñíûõ ãåíîâ ñ ïîìîùüþ ýêçîãåííî ââåäåííûõ ñèíòå-òè÷åñêèõ àíòèâèðóñíûõ êèÐÍÊ, òàê è ñ êîìïåíñàöèåéäåðåãóëÿöèè êëåòî÷íûõ ìèêðîÐÍÊ ïðè ãðèïïîçíîé èí-ôåêöèè.

Ìåõàíèçì ÐÍÊ-èíòåðôåðåíöèèè áèîãåíåç ìàëûõ èíòåðôåðèðóþùèõ ÐÍÊ

Â íàñòîÿùåå âðåìÿ èçâåñòíû òðè îñíîâíûõ òèïà ìà-ëûõ èíòåðôåðèðóþùèõ ÐÍÊ — ìèêðîÐÍÊ, êîðîòêèå èí-òåðôåðèðóþùèå ÐÍÊ (êèÐÍÊ) è Piwi-âçàèìîäåéñòâóþ-ùèå ÐÍÊ (piÐÍÊ).

Â ñîìàòè÷åñêèõ êëåòêàõ ïîçâîíî÷íûõ îñíîâíûì òè-ïîì ýíäîãåííûõ êîðîòêèõ ÐÍÊ, îïîñðåäóþùèõ ÏÒÃÑ,ÿâëÿþòñÿ ìèêðîÐÍÊ. ÌèêðîÐÍÊ ïðåäñòàâëÿþò ñîáîé êî-ðîòêèå (18—25 ï. í.) ÐÍÊ, êîäèðóåìûå ÿäåðíîé ÄÍÊ, àòàêæå ãåíîìàìè ìíîãèõ âèðóñîâ. Êëåòî÷íàÿ áåëêîâàÿ ìà-øèíåðèÿ, èñïîëüçóåìàÿ äëÿ ñèíòåçà è ïðîöåññèíãà ìèê-ðîÐÍÊ, ê íàñòîÿùåìó âðåìåíè äîñòàòî÷íî õîðîøî èçâå-ñòíà (Carthew, Sontheimer, 2009; Hammond, 2015). Ôîðìè-ðîâàíèå çðåëûõ ìèêðîÐÍÊ â êëåòêå ïðîèñõîäèò â

516 À. Í. Ãîðøêîâ è äð.

íåñêîëüêî ñòàäèé. Ïåðâè÷íûé ìèêðîÐÍÊ-òðàíñêðèïò(pri-miRNA), òðàíñêðèáèðóåìûé îáû÷íî ÐÍÊ-ïîëèìåðà-çîé 2 (Lee et al., 2004), èìååò äëèíó îò íåñêîëüêèõ ñîòåíäî íåñêîëüêèõ äåñÿòêîâ òûñÿ÷ íóêëåîòèäîâ (Saini et al.,2007). Ìîëåêóëû pri-miRNA ñîäåðæàò îäíó èëè íåñêîëü-êî øïèëå÷íûõ ñòðóêòóð, êîòîðûå â äàëüíåéøåì ñòàíî-âÿòñÿ èñòî÷íèêàìè îòäåëüíûõ ìèêðîÐÍÊ (Lee et al.,2002). Ïðîöåññèíã pri-miRNA ñâÿçàí ñ åå ýíäîíóêëåàç-íûì ðàñùåïëåíèåì. Ïåðâàÿ ôàçà ðàñùåïëåíèÿ pri-miRNAïðîèñõîäèò â ÿäðå è îñóùåñòâëÿåòñÿ ñ ïîìîùüþ áåëêîâî-ãî êîìïëåêñà, íàçûâàåìîãî ìèêðîïðîöåññîðîì, ñîñòîÿ-ùåãî èç ÐÍÊàçà III-ïîäîáíîãî ôåðìåíòà Drosha èÐÍÊ-ñâÿçûâàþùåãî áåëêà DGCR8. DGCR8 óçíàåò ôëàí-êèðóþùèå îäíîíèòåâûå áàçàëüíûå ó÷àñòêè â øïèëå÷íûõñòðóêòóðàõ pri-miRNA è îïðåäåëÿåò ïðàâèëüíóþ ëîêàëè-çàöèþ êîìïëåêñà. Drosha ðàçðåçàåò äâóõöåïî÷å÷íóþ ÐÍÊøïèëüêè íà ðàññòîÿíèè 11 ï. í. îò íà÷àëà ñòåáëÿ (Gregoryet al., 2004; Han et al., 2006). Ðèáîíóêëåàçíàÿ àêòèâíîñòüDrosha ïðèâîäèò ê âûñâîáîæäåíèþ ïðåäøåñòâåííèêàìèêðîÐÍÊ (pre-miRNA) äëèíîé 60—100 íóêëåîòèäîâ,èìåþùåãî âòîðè÷íóþ ñòðóêòóðó «ñòåáåëü—ïåòëÿ» (Zenget al., 2005). Ñóùåñòâóåò òàêæå àëüòåðíàòèâíûé ïóòü áèî-ãåíåçà pre-miRNA, ñâÿçàííûé ñî ñïëàéñèíãîì ìèê-ðîÐÍÊ-êîäèðóþùèõ èíòðîíîâ (ìèðòðîíîâ) è íå âîâëåêà-þùèé êîìïëåêñà ìèêðîïðîöåññîðà (Ruby et al., 2007).

Pre-miRNA ýêñïîðòèðóþòñÿ â öèòîïëàçìó ïðè ó÷àñ-òèè ýêñïîðòèíà-5, àññîöèèðîâàííîãî ñ ìàëîé ÃÒÔàçîéRan (Yi et al., 2003). Íà âòîðîì ýòàïå ýíäîíóêëåàçíîãî ïðî-öåññèíãà ÐÍÊàçà III Dicer óäàëÿåò ïåòëåâóþ îáëàñòü, îñòàâ-ëÿÿ ÐÍÊ-äóïëåêñ äëèíîé 21—23 ï. í. (Hutvagner et al.,2001). Óçíàâàíèå è ñâÿçûâàíèå Dicer ñ pre-miRNA ïðîèñõî-äÿò ïðè ó÷àñòèè áåëêà TRBP (Chendrimada et al., 2005).

Ïîñëå îòðåçàíèÿ ïåòëè ìèêðîÐÍÊ-äóïëåêñ ñâÿçûâà-åòñÿ ñ áåëêàìè ñåìåéñòâà Argonaut (Ago), ôîðìèðóÿ êîì-ïëåêñ RISC (ÐÍÊ-èíäóöèðîâàííûé êîìïëåêñ ñàéëåíñèí-ãà) (Peters, Meister, 2007). Ñòàäèÿ àññîöèàöèè Ago ñ ìèê-ðîÐÍÊ-äóïëåêñîì ÿâëÿåòñÿ êðàòêîâðåìåííîé, òàê êàêN-äîìåí áåëêîâ Ago îáëàäàåò ãåëèêàçíîé àêòèâíîñòüþ èîáåñïå÷èâàåò áûñòðîå ðàçìàòûâàíèå äâóõíèòåâîé ìèê-ðîÐÍÊ (Kwak, Tomari, 2012). Ïîñëå ðàçäåëåíèÿ îäíà èçíèòåé («ïàññàæèðñêàÿ íèòü») óäàëÿåòñÿ èç êîìïëåêñà, àäðóãàÿ («âåäóùàÿ íèòü») â êîìïëåêñå ñ Ago îïîñðåäóåòñàéëåíñèíã öåëåâûõ ãåíîâ.

Ñâÿçûâàíèå ìèêðîÐÍÊ â ñîñòàâå êîìïëåêñà RISC ñöåëåâûìè ìÐÍÊ îñóùåñòâëÿåòñÿ ñ íåïîëíîé êîìïëåìåí-òàðíîñòüþ. Óçíàâàíèå ìÐÍÊ-ìèøåíåé îáû÷íî ïðîèñõî-äèò çà ñ÷åò êîìïëåìåíòàðíîãî ñïàðèâàíèÿ íóêëåîòèäîâ2—7 íà 5R-êîíöå ìèêðîÐÍÊ (seed region) ñ 3R-íåòðàíñëèðó-åìîé îáëàñòüþ (3R-UTR) ìÐÍÊ (Bartel, 2009). Âî ìíîãèõìèêðîÐÍÊ èìåþòñÿ òàêæå äîïîëíèòåëüíûå ñàéòû êîìïëå-ìåíòàðíîñòè ïîìèìî seed region (Grimson et al., 2007).

ÏÒÃÑ äîñòèãàåòñÿ íåñêîëüêèìè âîçìîæíûìè ìåõà-íèçìàìè. Îäíèì èç íèõ ÿâëÿåòñÿ ðàçðåçàíèå öåëåâîéìÐÍÊ â ðåçóëüòàòå ýíäîíóêëåàçíîé àêòèâíîñòè Ago2 (Liuet al., 2004). Ñàéò ðàñùåïëåíèÿ ëîêàëèçîâàí ìåæäó 9-ì è10-ì íóêëåîòèäàìè c 5R-êîíöà ìèêðîÐÍÊ (Martinez et al.,2002). Èìåííî êîìïëåìåíòàðíîå ñïàðèâàíèå öåíòðàëü-íîé îáëàñòè ìèêðîÐÍÊ (9—11-é íóêëåîòèäû) ñ ìÐÍÊ ÿâ-ëÿåòñÿ íåîáõîäèìûì óñëîâèåì ýíäîíóêëåàçíîé àêòèâíî-ñòè Ago2 (Doench et al., 2003). Àëüòåðíàòèâíî RISC ìî-æåò èíèöèèðîâàòü äåàäåíèëèðîâàíèå öåëåâîé ìÐÍÊ è ååïîñëåäóþùóþ äåãðàäàöèþ (Wu et al., 2006) ïóòåì ðåêðó-òèðîâàíèÿ àäàïòåðíîãî áåëêà GW182 è äåàäåíèëàçíîãîêîìïëåêñà Ccr4—Not (Fabian et al., 2012). GW182 ñâÿçû-âàåò, êðîìå òîãî, áåëîê PABP. Ïðåäïîëîæèòåëüíî âçàè-

ìîäåéñòâèå GW182 ñ PABP äåëàåò íåâîçìîæíîéöèðêóëÿðèçàöèþ ìÐÍÊ, íåîáõîäèìóþ äëÿ èíèöèàöèèòðàíñëÿöèè áåëêà (Zekri et al., 2009). Ñõåìà áèîãåíåçà èîñíîâíûõ ýôôåêòîðíûõ ìåõàíèçìîâ äåéñòâèÿ ìèêðîÐÍÊïðåäñòàâëåíà íà ðèñóíêå.

Èìååòñÿ ðÿä äðóãèõ áåëêîâûõ âçàèìîäåéñòâèé, âî-âëåêàþùèõ êîìïëåêñ RISC. Â îáùåé ñëîæíîñòè ïðåäïî-ëàãàåòñÿ 9 âçàèìîäîïîëíÿþùèõ ìîëåêóëÿðíûõ ìåõàíèç-ìîâ RISC-îïîñðåäîâàííîãî ñàéëåíñèíãà ãåíîâ, äåòàëè êî-òîðûõ ïîêà äî êîíöà íå âûÿñíåíû (Morozova et al., 2012).

Â êëåòêå êàæäàÿ ìèêðîÐÍÊ ñïîñîáíà óçíàâàòü ðÿäìÐÍÊ-ìèøåíåé. Â òî æå âðåìÿ îäíà ìÐÍÊ ìîæåò ÿâëÿòü-ñÿ ìèøåíüþ äëÿ ìíîãèõ ìèêðîÐÍÊ (Krek et al., 2005).Â ðåçóëüòàòå ñîâîêóïíîñòü ìèêðîÐÍÊ îñóùåñòâëÿåòñëîæíóþ ñåòü âçàèìîäåéñòâèé c ìÐÍÊ-ìèøåíÿìè, îáåñ-ïå÷èâàÿ òîíêóþ íàñòðîéêó àêòèâíîñòè ãåíîâ è ðåãóëèðóÿâàæíåéøèå ôóíêöèè â íîðìå è ïðè ìíîæåñòâå ïàòîëîãè-÷åñêèõ ïðîöåññîâ (Ghildiyal, Zamore, 2009; Ðóêøà è äð.,2016). Ïî ðàçíûì îöåíêàì, 30—60 % ãåíîâ ÷åëîâåêà, êî-äèðóþùèõ áåëîê, íàõîäÿòñÿ ïîä ðåïðåññèâíûì êîíòðî-ëåì ìèêðîÐÍÊ (Lewis et al., 2005; Friedman et al., 2009).Êðîìå òîãî, ìíîãèå ìèêðîÐÍÊ â êëåòêå ñóùåñòâóþò â íå-ñêîëüêèõ èçîôîðìàõ (Guo, Chen, 2014).

Èíôåêöèÿ ÂÃÀ ñóùåñòâåííî èçìåíÿåò ïðîôèëü êëå-òî÷íûõ ìèêðîÐÍÊ, ïðè ýòîì íåêîòîðûå èçìåíåíèÿ ìîæ-íî îöåíèòü êàê ïðîâèðóñíûå, à íåêîòîðûå — êàê àíòèâè-ðóñíûå. Äèíàìèêà ìèêðîÐÍÊ ïðè ãðèïïå è åå áèîëîãè÷å-ñêàÿ ðîëü ïîäðîáíî ðàññìàòðèâàþòñÿ íèæå.

Âòîðûì òèïîì ìàëûõ èíòåðôåðèðóþùèõ ÐÍÊ ÿâëÿ-þòñÿ êèÐÍÊ. Ñòðóêòóðíî êèÐÍÊ ïðåäñòàâëÿþò ñîáîéäóïëåêñû äëèíîé 21—25 ï. í., ñõîæèå ñ âûøåîïèñàííû-ìè ìèêðîÐÍÊ-äóïëåêñàìè. Â îòëè÷èå îò ìèêðîÐÍÊêèÐÍÊ íå êîäèðóþòñÿ êëåòî÷íûìè ãåíàìè. Îíè áûëè îò-êðûòû â èññëåäîâàíèÿõ íà íåìàòîäå Caenorhabditis ele-gans êàê ïðîäóêò ðàçðåçàíèÿ ÐÍÊàçîé Dicer ýêçîãåííîââåäåííûõ â êëåòêè äëèííûõ äâóõíèòåâûõ ÐÍÊ (äíÐÍÊ),êîìïëåìåíòàðíûõ ê ýêçîííûì ó÷àñòêàì ãåíîâ (Fire et al.,1998). Â ðÿäå ãðóïï îðãàíèçìîâ èñòî÷íèêàìè êèÐÍÊ ïî-ìèìî ýêñïåðèìåíòàëüíî ââåäåííûõ ìîãóò áûòü òàêæå âè-ðóñíûå äíÐÍÊ è ïðîäóêòû äâóíàïðàâëåííîé òðàíñêðèï-öèè ìîáèëüíûõ ýëåìåíòîâ ãåíîìà. Ýôôåêòîðíûé ìåõà-íèçì êèÐÍÊ-èíòåðôåðåíöèè ñõîæ ñ âûøåîïèñàííûìãåííûì ñàéëåíñèíãîì, îïîñðåäîâàííûì ìèêðîÐÍÊ. Îíâêëþ÷àåò â ñåáÿ ñáîðêó êîìïëåêñà RISC è ôðàãìåíòàöèþìÐÍÊ-ìèøåíè áåëêîì Ago2.

Â ïîñëåäíèå ãîäû ñèíòåòè÷åñêèå êèÐÍÊ, íàïðàâëåí-íûå íà ïîäàâëåíèå ýêñïðåññèè ãåíîâ ÂÃÀ, àêòèâíî èññëå-äóþòñÿ â êà÷åñòâå ïåðñïåêòèâíûõ íîâûõ ëåêàðñòâåííûõñðåäñòâ â ëå÷åíèè ãðèïïà. Â ýòîì íàïðàâëåíèè â ðÿäå ëà-áîðàòîðèé äîñòèãíóòû çàìåòíûå óñïåõè, ðåçóëüòàòû äàí-íûõ ðàáîò äåòàëüíî îáñóæäàþòñÿ íèæå.

Òðåòüèì òèïîì ìàëûõ èíòåðôåðèðóþùèõ ÐÍÊ ÿâëÿ-þòñÿ piÐÍÊ, èìåþùèå äëèíó 25—32 íóêëåîòèäà. Îíèâçàèìîäåéñòâóþò ñ áåëêàìè êëàäà PIWI, òàêæå âõîäÿùè-ìè â ñåìåéñòâî Argonaut. Ó ìëåêîïèòàþùèõ áåëêè PIWIè piÐÍÊ îáíàðóæåíû òîëüêî â ãîíàäàõ. Áèîãåíåç piÐÍÊñóùåñòâåííî îòëè÷àåòñÿ îò áèîãåíåçà ìèðîÐÍÊ è êèÐÍÊ.piÐÍÊ ÷àñòè÷íî ïðîèñõîäÿò èç ñïåöèàëèçèðîâàííûõpiÐÍÊ-êëàñòåðîâ äëèíîé îò 1000 äî 100 000 ï. í. è áîëåå,÷àñòè÷íî — èç òðàíñêðèïòîâ àêòèâíûõ ìîáèëüíûõ ýëå-ìåíòîâ ãåíîìà. Ïî èìåþùèìñÿ äàííûì, ïðåäøåñòâåííè-êè piÐÍÊ â îòëè÷èå îò ïðåäøåñòâåííèêîâ ìèêðîÐÍÊ èêèÐÍÊ íå èìåþò øïèëå÷íîé ñòðóêòóðû è ÿâëÿþòñÿ îäíî-íèòåâûìè ìîëåêóëàìè. Îñíîâíîé ôóíêöèåé piÐÍÊ ÿâëÿ-åòñÿ ðåïðåññèÿ ïîäâèæíûõ ýëåìåíòîâ ãåíîìà (òðàíñïîçî-

ÐÍÊ-èíòåðôåðåíöèÿ è ïàòîãåíåç âèðóñà ãðèïïà À 517

íîâ) â êëåòêàõ çàðîäûøåâîãî ïóòè. Ýòà çàäà÷à ðåøàåòñÿêàê â ÿäðå, ãäå áåëêè PIWI âîâëå÷åíû â ðåãóëÿöèþ ñòðóê-òóðû õðîìàòèíà, ïðåïÿòñòâóþùóþ òðàíñêðèïöèè òðàíñ-ïîçîíîâ, òàê è â öèòîïëàçìå, ãäå êîìïëåêñû PIWI èpiÐÍÊ îáåñïå÷èâàþò êîìïëåìåíòàðíîå óçíàâàíèå è ôðàã-ìåíòàöèþ òðàíñêðèïòîâ ïîäâèæíûõ ýëåìåíòîâ (Tothet al., 2016).

piÐÍÊ, ïî-âèäèìîìó, íå âîâëå÷åíû â ïàòîãåíåç ÂÃÀ(ê íàñòîÿùåìó âðåìåíè èõ íå îáíàðóæåíî â ðåñïèðàòîð-íûõ ýïèòåëèÿõ), ïîýòîìó íèæå ìû ñêîíöåíòðèðóåìñÿ íàêèÐÍÊ- è ìèêðîÐÍÊ-âåòâÿõ ÐÍÊ-èíòåðôåðåíöèè.

Àíòèâèðóñíàÿ ÐÍÊ-èíòåðôåðåíöèÿíà îñíîâå êèÐÍÊ âèðóñíîãî ïðîèñõîæäåíèÿ

ÐÍÊ-èíòåðôåðåíöèÿ èãðàåò êëþ÷åâóþ ðîëü â ñëîæ-íûõ âçàèìîäåéñòâèÿõ ìåæäó âèðóñîì è èíôèöèðîâàííîéèì êëåòêîé. Ó ðàñòåíèé, ãðèáîâ è â íåêîòîðûõ ãðóïïàõ

áåñïîçâîíî÷íûõ (÷ëåíèñòîíîãèå è íåìàòîäû) ÐÍÊ-èíòåð-ôåðåíöèÿ íà îñíîâå êèÐÍÊ âèðóñíîãî ïðîèñõîæäåíèÿ ÿâ-ëÿåòñÿ âàæíåéøèì ìåõàíèçìîì ïðîòèâîâèðóñíîéçàùèòû. Âèðóñíûå äíÐÍÊ óçíàþòñÿ áåëêàìè ñåìåéñòâàDicer DCL1 (Caenorhabditis elegans), DCL2 (Drosophila),DCL3 è DCL4 (Arabidopsis thaliana), êîòîðûå ðàçðåçàþòèõ íà êîðîòêèå äâóõíèòåâûå âèðóñíûå êèÐÍÊ-ôðàãìåíòû(21—24 ï. í.), ñòðóêòóðíî ñõîæèå ñ âûøåîïèñàííûìèìèêðîÐÍÊ-äóïëåêñàìè (Ding, 2010). Ýòà ðèáîíóêëåàçíàÿàêòèâíîñòü ïåðå÷èñëåííûõ ôîðì Dicer, íàïðàâëåííàÿ íàâèðóñíûå äíÐÍÊ, ñàìà ïî ñåáå îáëàäàåò ïðîòèâîâèðóñ-íûì äåéñòâèåì. Îäíàêî ýôôåêòîðíûé èììóííûé ìåõà-íèçì ó äàííûõ îðãàíèçìîâ âêëþ÷àåò ñáîðêó êîìïëåêñàRISC, âêëþ÷àþùåãî âèðóñíûå êèÐÍÊ, óçíàâàíèå âèðóñ-íûõ ìÐÍÊ ñ ïîëíîé êîìïëåìåíòàðíîñòüþ è èõ ôðàãìåí-òàöèþ â ðåçóëüòàòå ýíäîíóêëåàçíîé àêòèâíîñòè áåëêîâAgo (Pantaleo et al., 2007; Schuck et al., 2013).

Âîïðîñ î òîì, íàñêîëüêî ðàñïðîñòðàíåíà è ôóíêöèî-íàëüíî çíà÷èìà àíòèâèðóñíàÿ êèÐÍÊ-èíòåðôåðåíöèÿ ó


Áèîãåíåç ìèêðîÐÍÊ è îñíîâíûå ýôôåêòîðíûå ìåõàíèçìû ïîñòòðàíñêðèïöèîííîãî ãåííîãî ñàéëåíñèíãà (ÏÒÃÑ).

Ïåðâè÷íûé ìèêðîÐÍÊ-òðàíñêðèïò (pri-miRNA) òðàíñêðèáèðóåòñÿ ÐÍÊ-ïîëèìåðàçîé 2 è ôîðìèðóåò øïèëå÷íóþ âòîðè÷íóþ ñòðóêòóðó. Äàëåå êîì-ïëåêñ ìèêðîïðîöåññîð (ÐÍÊàçà III-ïîäîáíûé ôåðìåíò Drosha è ÐÍÊ-ñâÿçûâàþùèé áåëîê DGCR8) âûðåçàåò ïðåäøåñòâåííèê ìèêðîÐÍÊ (pre-miRNA) ñîñòðóêòóðîé «ñòåáåëü—ïåòëÿ». Àëüòåðíàòèâíûé ïóòü áèîãåíåçà pre-miRNA ñâÿçàí ñî ñïëàéñèíãîì ìèêðîÐÍÊ-êîäèðóþùèõ èíòðîíîâ (ìèðòðîíîâ).Pre-miRNA ýêñïîðòèðóþòñÿ â öèòîïëàçìó ïðè ó÷àñòèè ýêñïîðòèíà 5 è ìàëîé ÃÒÔàçû Ran. Â öèòîïëàçìå ÐÍÊàçà III Dicer ïðè ó÷àñòèè áåëêà TRBPóäàëÿåò ïåòëåâóþ îáëàñòü, îñòàâëÿÿ ìèêðîÐÍÊ-äóïëåêñ äëèíîé 21—23 ï. í. ÌèêðîÐÍÊ-äóïëåêñ ñâÿçûâàåòñÿ ñ áåëêàìè ñåìåéñòâà Argonaut (Ago),ôîðìèðóÿ êîìïëåêñ RISC, ïðè ýòîì îäíà èç íèòåé (âåäóùàÿ íèòü) îïîñðåäóåò ÏÒÃÑ, à âòîðàÿ (ïàññàæèðñêàÿ) íèòü óäàëÿåòñÿ èç êîìïëåêñà. Âîçìîæ-íûå ýôôåêòîðíûå ìåõàíèçìû ÏÒÃÑ ñîñòîÿò â ðàçðåçàíèè öåëåâîé ìÐÍÊ áåëêîì Ago2, äåàäåíèëèðîâàíèè öåëåâîé ìÐÍÊ äåàäåíèëàçíûì êîìïëåêñîì

Ccr4—Not è ïîäàâëåíèè òðàíñëÿöèè ìÐÍÊ.

ìëåêîïèòàþùèõ, ÿâëÿåòñÿ äèñêóññèîííûì (Cullen et al.,2013; Cullen, 2014; Gantier, 2014; Weng et al., 2015). Â öå-ëîì õîðîøî èçâåñòíî, ÷òî ââåäåííûå â êëåòêè ìëåêîïèòà-þùèõ ýêçîãåííûå êèÐÍÊ âûçûâàþò âûñîêîýôôåêòèâíûéÏÒÃÑ öåëåâûõ ãåíîâ çà ñ÷åò ýíäîíóêëåàçíîé àêòèâíîñòèAgo2. Ñëåäîâàòåëüíî, ìëåêîïèòàþùèå îáëàäàþò íåîáõî-äèìûìè ýôôåêòîðíûìè ìåõàíèçìàìè äëÿ êèÐÍÊ-îïîñðå-äîâàííîé äåãðàäàöèè ìÐÍÊ-ìèøåíåé. Âìåñòå ñ òåì â îò-ëè÷èå îò ñåëåêòèâíîãî äåéñòâèÿ êèÐÍÊ òðàíñôåêöèÿ öå-ëåâûõ äëèííûõ äíÐÍÊ âûçûâàåò ëèøü íåñïåöèôè÷åñêîåïîäàâëåíèå ýêñïðåññèè ãåíîâ ïî ìåõàíèçìó èíòåðôåðî-íîâîãî îòâåòà (Elbashir et al., 2002), ò. å. â êëåòêàõ ìëåêî-ïèòàþùèõ äëèííûå äíÐÍÊ íå ïðîöåññèðóþòñÿ Dicer íàçíà÷èòåëüíîì óðîâíå â êèÐÍÊ-äóïëåêñû.

Áåññïîðíî, îñíîâíîé ìåõàíèçì àíòèâèðóñíîãî âðîæ-äåííîãî èììóíèòåòà ó ïîçâîíî÷íûõ — ýòî èíòåðôåðîíî-âûé îòâåò, â õîäå êîòîðîãî ðàñïîçíàâàíèå âèðóñíûõäíÐÍÊ áåðóò íà ñåáÿ áåëêîâûå ïàòòåðíðàñïîçíàþùèå ðå-öåïòîðû (Berke et al., 2013; Del Toro Duany et al., 2015).Åäèíñòâåííàÿ ôîðìà Dicer, ýêñïðåññèðóåìàÿ ó ìëåêîïè-òàþùèõ, íàïðàâëåíà íà ïðîäóêöèþ ìèêðîÐÍÊ èç øïè-ëå÷íûõ pre-miRNA; ðèáîíóêëåàçíàÿ àêòèâíîñòü Dicerìëåêîïèòàþùèõ â îòíîøåíèè äëèííûõ äÖÐÍÊ íèçêà çàñ÷åò àóòîèíãèáèòîðíîé ðîëè åãî N-êîíöåâîãî äîìåíà, ãî-ìîëîãè÷íîãî DExD/H-box-ãåëèêàçàì (Ma et al., 2008).Ôåðìåíòàòèâíàÿ àêòèâíîñòü Dicer òàêæå êðèòè÷åñêè çà-âèñèìà îò ðÿäà ïàðòíåðîâ ïî ñâÿçûâàíèþ, îïðåäåëÿþùèõåãî êîíôîðìàöèîííîå ñîñòîÿíèå: áåëêè TRBP, ADAR1 è,âîçìîæíî, RHA àêòèâèðóþò Dicer (Robb, Rana, 2007;Chakravarthy et al., 2010; Ota et al., 2013), â òî âðåìÿ êàêáåëîê PACT, íàïðîòèâ, èíãèáèðóåò àêòèâíîñòü Dicer â îò-íîøåíèè äëèííûõ äíÐÍÊ (Lee et al., 2013a).

Â ñîãëàñèè ñ ïåðå÷èñëåííûìè ôàêòàìè ãëóáîêîåñåêâåíèðîâàíèå êëåòîê ìëåêîïèòàþùèõ, èíôèöèðîâàí-íûõ ðàçëè÷íûìè âèðóñàìè, íå îáíàðóæèëî â íèõ ñóùåñò-âåííûõ êîëè÷åñòâ ìàëûõ ÐÍÊ, êîòîðûå ÿâíî ñîîòâåòñòâî-âàëè áû âñåì êðèòåðèÿì âèðóñíûõ êèÐÍÊ (ðàçìåðó22 � 2 íóêëåîòèäà, âûñîêîìó óðîâíþ àêêóìóëÿöèè, ðàâ-íîìó ÷èñëó ñìûñëîâûõ è àíòèñìûñëîâûõ íèòåé, ñïåöè-ôè÷åñêîé àññîöèàöèè ñ Ago) (Backes et al., 2014; Bogerdet al., 2014). Êðîìå òîãî, ïðîäåìîíñòðèðîâàíî, ÷òî â êëå-òî÷íûõ ëèíèÿõ ñ îòñóòñòâóþùåé àêòèâíîñòüþ ãåíà Dicerðåïëèêàöèÿ ðÿäà ÐÍÊ- è ÄÍÊ-âèðóñîâ, â òîì ÷èñëå ÂÃÀ,èäåò ñ òîé æå ñêîðîñòüþ, ÷òî è â èíòàêòíûõ êëåòêàõ (Bo-gerd et al., 2014). Ýòè ðåçóëüòàòû ïðèâåëè ðÿä àâòîðîâ êçàêëþ÷åíèþ, ÷òî êèÐÍÊ-èíòåðôåðåíöèÿ êàê ñðåäñòâîôóíêöèîíàëüíîé àíòèâèðóñíîé çàùèòû â ïðèíöèïå íåõà-ðàêòåðíà äëÿ ìëåêîïèòàþùèõ (Cullen et al., 2013; Backeset al., 2014; Bogerd et al., 2014).

Òåì íå ìåíåå ñóùåñòâóþò àðãóìåíòû è ïðîòèâîïî-ëîæíîãî ðîäà. Îáíàðóæåíî (Parameswaran et al., 2010), ÷òîêëåòêè ìëåêîïèòàþùèõ, èíôèöèðîâàííûå øåñòüþ ðàçëè÷-íûìè ÐÍÊ-âèðóñàìè, ñîäåðæàò êîðîòêèå ÐÍÊ âèðóñíîãîïðîèñõîæäåíèÿ, ÷àñòü èç êîòîðûõ ÿâëÿþòñÿ ÐÍÊ-äóïëåê-ñàìè ñ íåñêîëüêèìè (äî òðåõ) íåñïàðåííûìè íóêëåîòèäà-ìè íà 3R-êîíöàõ (õàðàêòåðíûé ïðèçíàê ýíäîíóêëåàçíîãîðàñùåïëåíèÿ Dicer). Äëÿ âèðóñà ãåïàòèòà Ñ àâòîðû ïðî-äåìîíñòðèðîâàëè òàêæå àññîöèàöèþ êîðîòêèõ âèðóñíûõÐÍÊ ñ îâåðýêñïðåññèðîâàííûìè Ago1—Ago4, õîòÿ èõ ðå-àëüíàÿ ôóíêöèîíàëüíàÿ ðîëü â ÐÍÊ-èíòåðôåðåíöèè âýòîé ðàáîòå íå áûëà ïîêàçàíà (Parameswaran et al., 2010).

Dicer-çàâèñèìàÿ ïðîäóêöèÿ êèÐÍÊ âèðóñíîãî ïðîèñ-õîæäåíèÿ è ñáîðêà êîìïëåêñà RISC áûëè îáíàðóæåíû âïëþðèïîòåíòíûõ ýìáðèîíàëüíûõ ñòâîëîâûõ êëåòêàõ(ÝÑÊ) ìûøè, èíôèöèðîâàííûõ âèðóñîì ýíöåôàëîìèî-

êàðäèòà (Picornaviridae) (Maillard et al., 2013). Äèôôåðåí-öèðîâêà ìûøèíûõ ÝÑÊ ðåçêî (â 10 ðàç) ñíèæàëà óðîâåíüâèðóñíîé êèÐÍÊ, ÷òî, ïî-âèäèìîìó, ñâÿçàíî ñ àêòèâà-öèåé ãåíîâ ñèñòåìû âðîæäåííîãî èììóíèòåòà (Flemret al., 2013). Óñòàíîâëåíî, ÷òî îîöèòû ìûøè ýêñïðåññè-ðóþò âûñîêîàêòèâíóþ N-òåðìèíàëüíî óêîðî÷åííóþ ôîð-ìó Dicer (Dicer0) ñ îòñóòñòâóþùèì DExD/H-box-ïîäîá-íûì äîìåíîì (Flemr et al., 2013); âîçìîæíî, Dicer0 ýêñ-ïðåññèðóåòñÿ è â ÝÑÊ ìûøè (Cullen, 2014). Óðîâåíüýêñïðåññèè Dicer â ÝÑÊ â öåëîì ñóùåñòâåííî âûøå, ÷åìâ äèôôåðåíöèðîâàííûõ ñîìàòè÷åñêèõ êëåòêàõ (Billyet al., 2001; Chen et al., 2010), ÷òî, î÷åâèäíî, òàêæå ñïî-ñîáñòâóåò áîëåå âûñîêîìó óðîâíþ ïðîòèâîâèðóñíîéêèÐÍÊ-èíòåðôåðåíöèè â ÝÑÊ.

Íåêîòîðîå óñèëåíèå ðåïëèêàöèè ÂÃÀ ïîñëå íîêäàóíàãåíà Dicer áûëî îáíàðóæåíî â êëåòî÷íîé ëèíèè Vero, õà-ðàêòåðèçóþùåéñÿ îòñóòñòâèåì ýêñïðåññèè ãåíîâ IFN-A1è IFN-B1 (Matskevich et al., 2007). Ýòè ðåçóëüòàòû ïîç-âîëÿþò ïðåäïîëîæèòü âîçìîæíîñòü àíòèâèðóñíîé êèÐÍÊ-èíòåðôåðåöèè â äàííîé èíòåðôåðîí-äåôèöèòíîé êëåòî÷-íîé ñèñòåìå è ïîäòâåðæäàþò, ÷òî â êëåòêàõ ìëåêîïèòàþ-ùèõ ñóùåñòâóþò êîíêóðåíòíûå îòíîøåíèÿ ìåæäó ñèñòå-ìîé èíòåðôåðîíîâîãî îòâåòà è àíòèâèðóñíîé êèÐÍÊ-èí-òåðôåðåíöèåé.

Ìíîãèå âèðóñû êîäèðóþò áåëêè-ñóïðåññîðû àíòèâè-ðóñíîé êèÐÍÊ-èíòåðôåðåíöèè, îãðàíè÷èâàþùèå ñïîñîá-íîñòü Dicer ðàçðåçàòü âèðóñíûå äíÐÍÊ. Çàðàæåíèå êëåòîêìëåêîïèòàþùèõ âèðóñàìè ñ äåëåöèÿìè èëè ìóòàöèÿìèñîîòâåòñòâóþùèõ ãåíîâ ïðèâîäèëî ê ïîÿâëåíèþ â íèõçíà÷èòåëüíûõ êîëè÷åñòâ âèðóñíûõ êèÐÍÊ è ê Ago2-îïî-ñðåäîâàííîìó ñíèæåíèþ èíôåêöèîííîé ñïîñîáíîñòè âè-ðóñà. Òàêèå ðåçóëüòàòû áûëè ïîëó÷åíû äëÿ âèðóñà Íîäà-ìóðà (Nodaviridae) ñ äåëåöèåé áåëêà B2 (NoDB2) (Li et al.,2013; Maillard et al., 2013), äëÿ âèðóñà ãåïàòèòà ìûøåé(Coronaviridae) c ìóòàöèåé áåëêà N (Cui et al., 2015). Íàìîäåëüíûõ ñèñòåìàõ ïîäàâëåíèå ýêñïåðèìåíòàëüíîéêèÐÍÊ-èíòåðôåðåíöèè áûëî ïðîäåìîíñòðèðîâàíî òàêæåäëÿ öåëîãî ðÿäà îâåðýêñïðåññèðîâàííûõ âèðóñíûõ ñó-ïðåññîðíûõ áåëêîâ, â òîì ÷èñëå NS1 èç ÂÃÀ (Bucheret al., 2004; Li et al., 2004). Äîêàçàíî ïðÿìîå ñâÿçûâàíèåäíÐÍÊ ñ N-êîíöåâûì (àìèíîêèñëîòíûå îñòàòêè 1—73) äî-ìåíîì áåëêà NS1 ÂÃÀ (Chien et al., 2004; Cheng et al.,2009), ïðåïÿòñòâóþùåå ðèáîíóêëåàçíîé àêòèâíîñòè Dicer.

Òàêèì îáðàçîì, ó ìëåêîïèòàþùèõ àíòèâèðóñíàÿêèÐÍÊ-èíòåðôåðåíöèÿ, ïî-âèäèìîìó, îãðàíè÷åíà îîöè-òàìè è ÝÑÊ (ïðåäïîëîæèòåëüíî òîëüêî ãðûçóíîâ), ÷òîìîæåò áûòü ñâÿçàíî ñ ýêñïðåññèåé â äàííûõ êëåòêàõ âû-ñîêîàêòèâíîãî Dicer0 (Flemr et al., 2013) è îòñóòñòâèåì âíèõ ñóùåñòâåííîãî óðîâíÿ ýêñïðåññèè ãåíîâ èíòåðôåðî-íîâîãî îòâåòà (Harada et al., 1990). Â äèôôåðåíöèðîâàí-íûõ ñîìàòè÷åñêèõ êëåòêàõ ìëåêîïèòàþùèõ êèÐÍÊ-èí-òåðôåðåíöèÿ, î÷åâèäíî, íå èãðàåò çàìåòíîé ðîëè â áîðüáåñ âèðóñàìè èç-çà âêëþ÷åíèÿ â êëåòêàõ ãåíîâ èíòåðôåðî-íîâîãî îòâåòà, à òàêæå ñóïðåññèè äíÐÍÊ-ïðîöåññèðóþùåéñïîñîáíîñòè Dicer ïî íåñêîëüêèì ìåõàíèçìàì (àóòîèíãè-áèðîâàíèå, èíãèáèðîâàíèå êëåòî÷íûìè áåëêàìè è èíãè-áèðîâàíèå ñóïðåññîðàìè âèðóñíîãî ïðîèñõîæäåíèÿ).

êèÐÍÊ-èíòåðôåðåíöèÿ êàê íîâûéòåðàïåâòè÷åñêèé ïîäõîä â áîðüáå ñ ÂÃÀ

Òåðàïåâòè÷åñêîå ïðèìåíåíèå êèÐÍÊ-èíòåðôåðåíöèèäëÿ ëå÷åíèÿ ðÿäà çàáîëåâàíèé, â òîì ÷èñëå ãðèïïà, àêòèâ-íî èññëåäóåòñÿ ñ íà÷àëà 2000-õ ãîäîâ. Â öåëîì óñïåõ


êèÐÍÊ-îïîñðåäîâàííîãî ñàéëåíñèíãà öåëåâîãî ãåíà ïîñóùåñòâó îïðåäåëÿåòñÿ äâóìÿ êîìïîíåíòàìè — îïòè-ìàëüíûì äèçàéíîì êèÐÍÊ è âûáîðîì ýôôåêòèâíîãîñðåäñòâà äîñòàâêè êèÐÍÊ â êëåòêè-ìèøåíè.

Â ðÿäå ðàáîò ýêñïåðèìåíòàëüíî áûëè îïðåäåëåíûáëàãîïðèÿòíûå è íåáëàãîïðèÿòíûå ôàêòîðû äèçàéíàêèÐÍÊ, âëèÿþùèå íà ôèíàëüíóþ ýôôåêòèâíîñòü è ñïå-öèôè÷íîñòü êèÐÍÊ-èíòåðôåðåíöèè (Reynolds et al., 2004;Birmingham et al., 2007). Áûëè ðàçðàáîòàíû àëãîðèòìûïîäáîðà êèÐÍÊ, ó÷èòûâàþùèå ïåðå÷èñëåííûå âûøå òðå-áîâàíèÿ ê èõ äèçàéíó (Birmingham et al., 2007; Wang et al.,2009). Ñóùåñòâóþò web-ðåñóðñû, íàöåëåííûå íà ïîäáîðêèÐÍÊ äëÿ ñàéëåíñèíãà öåëåâûõ ãåíîâ (Lagana et al.,2015). Â íàñòîÿùåå âðåìÿ äîñòóïíû ïîëíîãåíîìíûå áèá-ëèîòåêè êèÐÍÊ, ïîçâîëÿþùèå ïðîâîäèòü ðàçëè÷íûå âû-ñîêîïðîèçâîäèòåëüíûå ôóíêöèîíàëüíûå èññëåäîâàíèÿ íàîñíîâå íîêäàóíîâ èíäèâèäóàëüíûõ ãåíîâ. Â èññëåäîâàíè-ÿõ in vitro íàðÿäó ñ ñîáñòâåííî êèÐÍÊ ÷àñòî èñïîëüçóþò-ñÿ ïëàçìèäû èëè ëåíòèâèðóñíûå âåêòîðû, êîäèðóþùèåøïèëå÷íûå ïðåäøåñòâåííèêè êèÐÍÊ (êøÐÍÊ).

Âòîðûì êðèòè÷åñêè âàæíûì îáñòîÿòåëüñòâîì, îïðå-äåëÿþùèì óñïåøíîñòü êèÐÍÊ-èíòåðôåðåíöèè è â ïåð-ñïåêòèâå êèÐÍÊ-òåðàïèè, ÿâëÿåòñÿ ñîçäàíèå ñðåäñòâà äî-ñòàâêè êèÐÍÊ, êîòîðîå îäíîâðåìåííî ñîîòâåòñòâîâàëîáû öåëîìó ðÿäó íåîáõîäèìûõ òðåáîâàíèé — âûñîêîé ýô-ôåêòèâíîñòè äîñòàâêè êèÐÍÊ â öåëåâûå êëåòêè (â ñëó÷àåãðèïïà — â êëåòêè ðåñïèðàòîðíûõ ýïèòåëèåâ), çàùèòåêèÐÍÊ îò íóêëåàç âî âíåêëåòî÷íîé ñðåäå, íèçêîé òîêñè÷-íîñòè, îòñóòñòâèþ èììóíîãåííîñòè, ýôôåêòèâíîé áèî-ðàçëàãàåìîñòè è âûâåäåíèþ èç îðãàíèçìà. Â ñëó÷àå ñèñ-òåìíîãî ââåäåíèÿ ê òðåáîâàíèÿì îòíîñèòñÿ ñïîñîáíîñòüïðåîäîëåâàòü ðÿä òêàíåâûõ áàðüåðîâ (ýíäîòåëèé, áàçàëü-íûå ìåìáðàíû, òêàíåâûå ìàêðîôàãè), à â ñëó÷àå ýíäîöè-òîçíîãî ïîãëîùåíèÿ êëåòêàìè — ñïîñîáíîñòü ê âûñâî-áîæäåíèþ èç ýíäîñîì. Ïðåäëîæåíî íåñêîëüêî ýêñïåðè-ìåíòàëüíûõ ïëàòôîðì äîñòàâêè êèÐÍÊ, îñíîâíûìè èçêîòîðûõ ÿâëÿþòñÿ ëèïîñîìû, âèðóñíûå âåêòîðû, ïîëèêà-òèîííûå íàíîíîñèòåëè è ïåïòèäíûå ñèñòåìû äîñòàâêè(Wang et al., 2010; Ruigrok et al., 2016). Íåñìîòðÿ íà áîëü-øîå êîëè÷åñòâî èññëåäîâàíèé â äàííîì íàïðàâëåíèè èðÿä îáíàäåæèâàþùèõ ðåçóëüòàòîâ, âîïðîñ âûáîðà îïòè-ìàëüíîé ñòðàòåãèè äîñòàâêè òåðàïåâòè÷åñêèõ êèÐÍÊin vivo íå ìîæåò ïîêà ñ÷èòàòüñÿ ðåøåííûì.

Ïîäàâëåíèå ðåïëèêàöèè ÂÃÀ ñ ïîìîùüþ êèÐÍÊ-èí-òåðôåðåíöèè âèðóñíûõ ãåíîâ íåîäíîêðàòíî èññëåäîâàëèýêñïåðèìåíòàëüíî. Êàê è äëÿ êëåòî÷íûõ ãåíîâ, äëÿ ãåíîâÂÃÀ áûëè ñôîðìóëèðîâàíû áèîèíôîðìàöèîííûå àëãîðèò-ìû ïîäáîðà íàèáîëåå ýôôåêòèâíî äåéñòâóþùèõ êèÐÍÊ(ElHefnawi et al., 2011; Liu et al., 2013); äëÿ ðåøåíèÿ ýòîéçàäà÷è èìååòñÿ ðÿä web-ðåñóðñîâ (Sharma et al., 2015).

Ðåçóëüòàòû ýòèõ èññëåäîâàíèé ñóììèðîâàíû âòàáë. 1. Èç ïðèâåäåííûõ â òàáëèöå äàííûõ î÷åâèäíî, ÷òîýôôåêòèâíî ôóíêöèîíèðóþùèå àíòè-ÂÃÀ êèÐÍÊ (èëèêøÐÍÊ-êîäèðóþùèå ïëàçìèäû), êàê ïðàâèëî, íàïðàâëå-íû íà êîíñåðâàòèâíûå âèðóñíûå ãåíû, êîäèðóþùèå âíóò-ðåííèå áåëêè âèðóñà è ïîëèìåðàçíûé êîìïëåêñ. Â ðå-çóëüòàòå òàêîãî âûáîðà ìèøåíåé êèÐÍÊ-èíòåðôåðåíöèèâ ðÿäå ñëó÷àåâ óäàëîñü äîñòè÷ü èíãèáèðîâàíèÿ íåñêîëü-êèõ øòàììîâ ÂÃÀ îäíèìè è òåìè æå êèÐÍÊ (òàáë. 1).

Êàê âèäíî èç äàííûõ òàáë. 1, â ýêñïåðèìåíòàõ ïî àí-òè-ÂÃÀ êèÐÍÊ-èíòåðôåðåíöèè áûëè èñïîëüçîâàíûðàçëè÷íûå ñðåäñòâà äîñòàâêè êèÐÍÊ — êàê òðàäèöèîí-íûå, òàê è äîñòàòî÷íî íîâûå — ÏÝÃèëèðîâàííûå èììó-íîëèïîñîìû (Khantasup et al., 2014) è pH-çàâèñèìûå ïåï-òèäû (Liang et al., 2015). Ïîñêîëüêó ÂÃÀ ÿâëÿåòñÿ ðåñ-

ïèðàòîðíûì âèðóñîì, in vivo ñóùåñòâóåò âîçìîæíîñòüèíòðàíàçàëüíîãî èëè èíãàëÿöèîííîãî ââåäåíèÿ êèÐÍÊ-ñîäåðæàùèõ ïðåïàðàòîâ, ÷òî ïîçâîëÿåò èçáåæàòü ðÿäà ñå-ðüåçíûõ ïðåïÿòñòâèé, ñâÿçàííûõ ñ èõ ñèñòåìíûì ââåäå-íèåì (Ruigrok et al., 2016). Òåì íå ìåíåå ïðîáëåìû çàùè-òû êèÐÍÊ îò íóêëåàç è âûñâîáîæäåíèÿ êèÐÍÊ èç ýíäî-ñîì ýïèòåëèàëüíûõ êëåòîê îñòàþòñÿ àêòóàëüíûìè è âýòîì ñëó÷àå. Íåñìîòðÿ íà ïðèâåäåííûå â òàáë. 1 ïîëîæè-òåëüíûå ðåçóëüòàòû, èìåííî ðàçðàáîòêà îïòèìàëüíîãîñðåäñòâà äîñòàâêè êèÐÍÊ in vivo â èíôèöèðîâàííûå ÂÃÀêëåòêè îñòàåòñÿ îñíîâíûì ïðåïÿòñòâèåì, çàòðóäíÿþùèìíåìåäëåííóþ òðàíñëÿöèþ êèÐÍÊ-òåõíîëîãèè â êëèíè÷å-ñêóþ ïðàêòèêó òåðàïèè ãðèïïà.

Åùå îäíèì ïåðñïåêòèâíûì íàïðàâëåíèåì èñïîëüçî-âàíèÿ êèÐÍÊ â áîðüáå ñ ãðèïïîì ÿâëÿåòñÿ ïîèñê è ÏÒÃÑíå âèðóñíûõ, à êëåòî÷íûõ ãåíîâ, âàæíûõ äëÿ ðåïëèêàöèèÂÃÀ. Â ðÿäå ðàáîò ñ ýòîé öåëüþ áûë ïðîâåäåí ñêðèíèíãêëåòî÷íûõ ôàêòîðîâ ðåïëèêàöèè ÂÃÀ ñ èñïîëüçîâàíèåìêèÐÍÊ-áèáëèîòåê. Ïîëíîãåíîìíûé êèÐÍÊ-ñêðèíèíã ÿâ-ëÿåòñÿ ìîùíåéøèì èíñòðóìåíòîì â ôóíêöèîíàëüíûõ ãå-íåòè÷åñêèõ èññëåäîâàíèÿõ áèîëîãèè ÂÃÀ, îäíàêî åãî ðå-çóëüòàòû îêàçûâàþòñÿ êðèòè÷åñêè çàâèñèìûìè îò âû-áðàííûõ â èññëåäîâàíèè ïàðàìåòðîâ — êëåòî÷íîé ëèíèè,øòàììà âèðóñà, ýôôåêòèâíîñòè êèÐÍÊ-íîêäàóíà, âûáîðàâðåìåíí*ûõ òî÷åê ñêðèíèíãà. Â ÷àñòíîñòè, â 7 ïîëíîãå-íîìíûõ èññëåäîâàíèÿõ ñ èñïîëüçîâàíèåì áèáëèîòåêêèÐÍÊ, ïðîâåäåííûõ ê íàñòîÿùåìó âðåìåíè, áûëî âûÿâ-ëåíî â îáùåé ñëîæíîñòè 1362 õîçÿéñêèõ ãåíà, ñâÿçàííûõñ ðåïëèêàöèåé ÂÃÀ. Îäíàêî òîëüêî 6 ãåíîâ (ATP6AP1,ATP6V0C, ATP6V0D1, COPA, COPG è NXF1) ïåðåñåêà-ëèñü â ÷åòûðåõ ñêðèíèíãàõ è, ïàðàäîêñàëüíûì îáðàçîì,íè îäíîãî ãåíà — âî âñåõ 7 èññëåäîâàíèÿõ (Chou et al.,2015). Ïîýòîìó èíòåðïðåòàöèÿ ðåçóëüòàòîâ êèÐÍÊ-ñêðè-íèíãà è îñîáåííî èõ ïîñëåäóþùèé ïåðåâîä â ïëîñêîñòüðàçðàáîòêè ëåêàðñòâåííûõ ñðåäñòâ òðåáóþò îïðåäåëåí-íîé îñòîðîæíîñòè ïðè âñåé âàæíîñòè è èíôîðìàòèâíîñòèòàêèõ äàííûõ.

Âèðóñíûå ìèêðîÐÍÊ:ïðîâèðóñíàÿ ìèêðîÐÍÊ-èíòåðôåðåíöèÿ

Ðÿä âèðóñîâ èñïîëüçóåò ÐÍÊ-èíòåðôåðåíöèþ äëÿîáåñïå÷åíèÿ áåñïðåïÿòñòâåííîãî ïðîõîæäåíèÿ èìè æèç-íåííîãî öèêëà, êîäèðóÿ â ñâîèõ ãåíîìàõ âèðóñíûå ìèê-ðîÐÍÊ. Âèðóñíûå ìèêðîÐÍÊ êîäèðóþòñÿ ãåíîìàìè ðÿäàÄÍÊ-ñîäåðæàùèõ âèðóñîâ, ïðèíàäëåæàùèõ ê íåñêîëü-êèì ñåìåéñòâàì: Herpesviridae, Polyomaviridae, Ascoviri-dae, Baculoviridae, Adenoviridae è Iridoviridae (Grundhoff,Sullivan, 2011; Takane, Kanai, 2011; Liu, 2014). Áîëåå200 âèðóñíûõ ìèêðîÐÍÊ áûëî îáíàðóæåíî â ãåíîìàõàëüôà-, áåòà- è ãàììà-ãåðïåñ-âèðóñîâ. Âèðóñíûå ìèê-ðîÐÍÊ ÄÍÊ-ñîäåðæàùèõ âèðóñîâ èìåþò ìíîæåñòâî ìè-øåíåé êàê ñðåäè âèðóñíûõ ìÐÍÊ (àóòîðåãóëÿöèÿ âèðóñ-íûõ ãåíîâ) (Murphy et al., 2008; Lin et al., 2011), òàê è ñðå-äè êëåòî÷íûõ ìÐÍÊ. Âèðóñíûå ìèêðîÐÍÊ ðåãóëèðóþòïåðåêëþ÷åíèå ñ ëèòè÷åñêîãî íà ïåðñèñòåíòíûé òèï èí-ôåêöèè, âûçûâàþò íåîáõîäèìûå âèðóñó èçìåíåíèÿ êëå-òî÷íîãî ìåòàáîëèçìà, ìîäóëèðóþò ïðîõîæäåíèå êëåòêîéêëåòî÷íîãî öèêëà, îáåñïå÷èâàþò óêëîíåíèå îò äåéñòâèÿèììóííîé ñèñòåìû õîçÿèíà, èíãèáèðóþò àïîïòîç èíôè-öèðîâàííûõ êëåòîê (Grundhoff, Sullivan, 2011; Takane,Kanai, 2011; Powdrill et al., 2016).

Äàííûå ïî êîäèðîâàíèþ ìèêðîÐÍÊ ÐÍÊ-ñîäåðæà-ùèìè âèðóñàìè ê íàñòîÿùåìó âðåìåíè î÷åíü ñêóäíû.



Ò à á ë è ö à 1

Ýêñïåðèìåíòàëüíîå èñïîëüçîâàíèå êèÐÍÊ, íàïðàâëåííûõ íà âèðóñíûå ìèøåíè,äëÿ ýôôåêòèâíîãî ïîäàâëåíèÿ ðåïëèêàöèè âèðóñà ãðèïïà

Ìîäåëü Èíãèáèðóåìûå øòàììû ÂÃÀ Ñèñòåìà äîñòàâêè êèÐÍÊÌèøåíè êèÐÍÊ-èíòåðôåðåíöèè

Ëèòåðàòóðíûéèñòî÷íèê

Êëåòêè 293T,BHK

A/WSN/33 (H1N1) ñ íóêëåîòèäíîéçàìåíîé âàëèíà íà àëàíèí â ïîçè-öèè 41 â ÎÐÑ M1

ÒÔÐ Trans IT LT-1 + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

M2 McCown et al., 2003

Êëåòêè MDCK A/Puerto Rico/8/34 (H1N1),A/WSN/33 (H1N1)

Ýëåêòðîïîðàöèÿ êèÐÍÊ NP, PA, PB1 Ge et al., 2003

Êëåòêè Vero A/Puerto Rico/8/34 (H1N1) NP

Êóðèíûå ýì-áðèîíû

ÒÔÐ Oligofectamine + êèÐÍÊ NP, PA, PB1

Ìûøè Òî æå ÏÝÈ + êèÐÍÊ (âíóòðèâåííîå ââå-äåíèå)

NP, PA Ge et al., 2004

ÏÝÈ + ïëàçìèäíàÿ ÄÍÊ, êîäè-ðóþùàÿ êøÐÍÊ (ÂÂ)

NP

ÏÝÈ + ïëàçìèäíàÿ ÄÍÊ, êîäè-ðóþùàÿ êøÐÍÊ (ÈÍ)

NP, PB1

Êëåòêè Vero Ëåíòèâèðóñíûé âåêòîð, êîäèðóþ-ùèé êøÐÍÊ

NP

Ìûøè A/Puerto Rico/8/34 (H1N1), Ñâîáîäíàÿ êèÐÍÊ (ÂÂ) ñ ïîñëå-äóþùèì ÒÔÐ Oligofectamine +êèÐÍÊ (ÈÍ)

NP, PA Tompkins et al., 2004

A/Hong Kong/156/97 (H5N1),

A/Netherlands/219/03 (H7N7),

A/Hong Kong/1073/99 (H9N2)

Êëåòêè MDCK A/WSN/33 (H1N1) Ëåíòèâèðóñíûé âåêòîð, êîäèðóþ-ùèé êøÐÍÊ

M1 Hui et al., 2004

Ìûøè A/PuertoRico/8/34 (H1N1) Äåàöåòèëèðîâàííûé ÏÝÈ + êèÐÍÊ,(ÂÂ)

NP Thomas et al., 2005

Êëåòêè MDCK A/chicken/Hubei/327/2004 (H5N1), ÒÔÐ Lipofectamine + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

NP, M2 Zhou et al., 2007

A/Duck/Hubei/W1/2004

Ìûøè (H9N2), A/Hubei/2003 (H1N1) ÏÝÈ + ïëàçìèäíàÿ ÄÍÊ, êîäèðóþ-ùàÿ êøÐÍÊ (ÂÂ)

Êëåòêè MDCK A/chicken/Qinghaihu/726/2005(H5N1)

ÒÔÐ SuperFect + ïëàçìèäíàÿ ÄÍÊ,êîäèðóþùàÿ êøÐÍÊ

NP, PA, PB1 Zhou et al., 2008


ÒÔÐ Oligofectamine + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

Ìûøè Ïëàçìèäíàÿ ÄÍÊ, êîäèðóþùàÿêøÐÍÊ (ÂÂ)

Êëåòêè CEF A/Chicken/Henan/1/04 (H5N1) ÒÔÐ Lipofectamine + ÐÍÊ-îëèãî-íóêëåîòèäû, ìîäèôèöèðîâàííûå2R-O-ìåòèë ñ 3R-áóòàíîë-ìåòêîé

NS1 Wu et al., 2008

Öûïëÿòà ÒÔÐ Lipofectamine + ÐÍÊ-îëèãî-íóêëåîòèäû, ìîäèôèöèðîâàííûå2R-O-ìåòèë ñ 3R-áóòàíîë-ìåòêîé(ÈÍ)

Êëåòêè CH-SAHè MDCK

A/turkey/Ontario/6118/68 (H8N4), ÒÔÐ Lipofectamine + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

NP, PA Abrahamyan et al.,2009A/turkey/Massachusetts/3740/65

(H6N2),

A/duck/Czech/56 (H4N6),

A/Quail/Italy/1117/65 (H10N8)

Êëåòêè MDCK A/New Caledonia/20/1999 (H1N1),A/Hong Kong/486/97 (H5N1)

ÒÔÐ Lipofectamine + êèÐÍÊ, ëåí-òèâèðóñíûé âåêòîð, êîäèðóþ-ùèé êøÐÍÊ

M2 Sui et al., 2009

Êëåòêè MDCK A/PuertoRico/8/34 (H1N1) Áàêóëîâèðóñíûé âåêòîð, êîäèðóþ-ùèé êøÐÍÊ

NP Suzuki et al., 2009

A/Tiger/HarBin/01/2002 (H5N1) ÒÔÐ Lipofectamine + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

PA Zhang et al., 2009

Ìûøè ÏÝÈ + ïëàçìèäíàÿ ÄÍÊ, êîäèðóþ-ùàÿ êøÐÍÊ (ÂÂ)

Ñ íàèáîëüøåé äîñòîâåðíîñòüþ ãåíû ìèêðîÐÍÊ áûëè îá-íàðóæåíû â ãåíîìàõ âèðóñîâ ñåìåéñòâà Retroviridae,âêëþ÷àÿ ÂÈ×-1 (Omoto et al., 2004; Zhang et al., 2014).Ðåòðîâèðóñû èìåþò â ñâîåì æèçíåííîì öèêëå ñòàäèþ îá-ðàòíîé òðàíñêðèïöèè è èíòåãðàöèè ïðîâèðóñíîé ÄÍÊ âãåíîì êëåòêè-õîçÿèíà, ÷òî ïîçâîëÿåò èì èñïîëüçîâàòü äëÿñèíòåçà è ïðîöåññèíãà ñîáñòâåííûõ ìèêðîÐÍÊ êëåòî÷-íóþ ìèêðîÐÍÊ-ïðîèçâîäÿùóþ áåëêîâóþ ìàøèíåðèþ(Harwig et al., 2014).

Ðÿä àâòîðîâ ïîëàãàåò, ÷òî ìèêðîÐÍÊ, âîçìîæíî,â ïðèíöèïå íåñâîéñòâåííû ÐÍÊ-âèðóñàì ïî äâóì ïðè-

÷èíàì. Âî-ïåðâûõ, ìíîãèå ÐÍÊ-âèðóñû èìåþò öèòî-ïëàçìàòè÷åñêèé öèêë ðåïëèêàöèè è âíóòðèÿäåðíûåêîìïîíåíòû ìèêðîÐÍÊ-ïðîöåññèíãà îêàçûâàþòñÿ äëÿíèõ íåäîñòóïíûìè. Âî-âòîðûõ, ýíäîíóêëåàçíîå ðàçðåçà-íèå ïåðâè÷íûõ ìèêðîÐÍÊ-òðàíñêðèïòîâ ñïîñîáíî äåñòà-áèëèçèðîâàòü âèðóñíûé ãåíîì è âûçâàòü åãî ôðàãìåíòà-öèþ (Cullen, 2010; Grundhoff, Sullivan, 2011).

Âìåñòå ñ òåì îïóáëèêîâàíû ðåçóëüòàòû ðàáîò ïî ãå-íåòè÷åñêîìó èíæèíèðèíãó ÂÃÀ (Varble et al., 2010; Varb-le, tenOever, 2011) âèðóñà êëåùåâîãî ýíöåôàëèòà (Flavivi-ridae) (Rouha et al., 2010) è âèðóñà Ñèíäáèñ (Togoviridae)


Ò à á ë è ö à 1 (ïðîäîëæåíèå)

Ìîäåëü Èíãèáèðóåìûå øòàììû ÂÃÀ Ñèñòåìà äîñòàâêè êèÐÍÊÌèøåíè êèÐÍÊ-èíòåðôåðåíöèè


Êëåòêè MDCK A/PuertoRico/8/34 (H1N1) ÒÔÐ Lipofectamine + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

PB1 Li et al., 2011


Ìûøè Òî æå ÒÔÐ Lipofectamine + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ(âíóòðèâåííîå ââåäåíèå)

ÏÝÈ + êèÐÍÊ (ÂÂ ñ ïîñëåäóþùèìÈÍ)

NS1 Rajput et al., 2012

Êëåòêè MDCK A/Chicken/Thailand/VSMU-3-BKK/2004 (H5N1),

Êàòèîííûå ÏÝÃèëèðîâàííûå èììó-íîëèïîñîìû + êèÐÍÊ

NP Khantasup et al., 2014

A/Openbillstork/Thailand/VSMU-5-NSN/2004 (H5N1)

Êëåòêè HD11 A/Puerto Rico/8/34 (H1N1) ÒÔÐ Lipofectamine + êèÐÍÊ,ABA-21/117Q ïîëèìåð + êèÐÍÊ

PB1 Hinton et al., 2014


ABA-21/117Q ïîëèìåð + êèÐÍÊ

Êëåòêè MDCK Òî æå ÒÔÐ TransPass HeLa + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ

NS1, NS2 Svancarova et al.,2015

Ìûøè » » ÒÔÐ TransPass HeLa + ïëàçìèäíàÿÄÍÊ, êîäèðóþùàÿ êøÐÍÊ (ÂÂ)

Êëåòêè MDCK A/turkey/Italy/2676/2000(H7N1),

ÒÔÐ Effectene + ïëàçìèäíàÿ ÄÍÊ,êîäèðóþùàÿ êøÐÍÊ

NP Stoppani et al., 2015

A/swine/Italy/1521/1998 (H1N2),



A/swine/Italy/437/1976 (H1N1)

A/chicken/Navapur/7972/2006(H5N1)

ÒÔÐ X-treme Gene Transfectant +êèÐÍÊ

NP, PB2 Behera et al., 2015

Êëåòêè MDCK,A549

A/Puerto Rico/8/34 (H1N1) pH-çàâèñèìûå ïåïòèäû + êèÐÍÊ NP Liang et al., 2015

Êëåòêè MDCK Òî æå Ëåíòèâèðóñíûé âåêòîð, êîäèðóþ-ùèé êøÐÍÊ

NP, PB1 Xu et al., 2015

A/WS/33(H1N1) ÒÔÐ Lipofectamine + êèÐÍÊ M1, M2, NS1,NS2

McMillen et al., 2016

Êëåòêè LMH A/chicken/Texas/473-2/2010(H6N2),

Ïëàçìèäíàÿ ÄÍÊ, êîäèðóþùàÿêøÐÍÊ, òðàíñôîðìèðîâàííàÿâ E. coli áàêòåðèàëüíûé âåêòîðtkRNAi

NP, PA Linke et al., 2016

A/turkey/Colorado/235497/2003(H8N4)

Êëåòêè A549 A/WSN/33 (H1N1), Ëåíòèâèðóñíûé âåêòîð, êîäèðóþ-ùèé êøÐÍÊ

Íåéðàìèíèäàçà Wang et al., 2016

A/Puerto Rico/8/34 (H1N1)

Òðàíñãåííûåìûøè, ýêñ-ïðåññèðóþ-ùèå êøÐÍÊ

Ï ð è ì å ÷ à í è å. ÒÔÐ — òðàíñôåêöèîííûé ðåàãåíò, ÏÝÈ — ïîëèýòèëåíèìèí, ÏÝÃ-ïîëèýòèëåíãëèêîëü, ÂÂ è ÈÍ — ñîîòâåòñòâåííî âíóòðèâåí-íî è èíòðîíîçàëüíî.

(Shapiro et al., 2010), äîêàçûâàþùèå âîçìîæíîñòü âñòàâêèøïèëå÷íîãî ìèêðîÐÍÊ-ïðåäøåñòâåííèêà â íåêîäèðóþ-ùóþ îáëàñòü âèðóñíîãî ÐÍÊ-ãåíîìà è ïîñëåäóþùóþýêñïðåññèþ äàííîé ôóíêöèîíàëüíîé ìèêðîÐÍÊ â èíôè-öèðîâàííûõ êëåòêàõ áåç óòðàòû ðåïëèêàòèâíûõ ñâîéñòââèðóñà. Ïîìèìî î÷åâèäíîé ôóíäàìåíòàëüíîé âàæíîñòèïðèâåäåííûõ âûøå ðàáîò îíè îòêðûâàþò ïåðñïåêòèâó èñ-ïîëüçîâàíèÿ ÐÍÊ-ñîäåðæàùèõ âèðóñîâ â êà÷åñòâå íîâûõâåêòîðîâ äëÿ äîñòàâêè öåëåâûõ òåðàïåâòè÷åñêèõ ìèê-ðîÐÍÊ â êëåòêè (tenOever, 2013).

Îñîáåííî èíòåðåñíî, ÷òî åñëè âèðóñ ãðèïïà èìååòâíóòðèÿäåðíóþ ôàçó æèçíåííîãî öèêëà, Flaviviridae è To-goviridae ñ÷èòàþòñÿ ñåìåéñòâàìè ÐÍÊ-âèðóñîâ ñ ïîë-íîñòüþ öèòîïëàçìàòè÷åñêèì öèêëîì ðåïëèêàöèè. Òåì íåìåíåå äëÿ ôëàâèâèðóñà ïðîöåññèíã ïðå-ìèêðîÐÍÊ òðàíñ-êðèïòà îêàçàëñÿ çàâèñèìûì îò ÿäåðíîé íóêëåàçû Drosha.Àâòîðû èññëåäîâàíèÿ îáúÿñíÿþò ýòîò íåîáû÷íûé ôàêòâîçìîæíûì ïåðåðàñïðåäåëåíèåì ïîòîêîâ âíóòðèêëåòî÷-íîãî òðàíñïîðòà ïðè ôëàâèâèðóñíîé èíôåêöèè (Rouhaet al., 2010). Ïîçäíåå òðàíñëîêàöèÿ Drosha â öèòîïëàçìóïðè èíôèöèðîâàíèè êëåòîê âèðóñîì Ñèíäáèñ áûëà äîêà-çàíà ýêñïåðèìåíòàëüíî (Shapiro et al., 2012). Ïî ìíåíèþàâòîðîâ ðàáîòû, ýòîò ñëó÷àé ïðåäñòàâëÿåò ñîáîé íîâûéïîëíîñòüþ öèòîïëàçìàòè÷åñêèé ïóòü ïðîöåññèíãà øïè-ëå÷íîé ÐÍÊ â êëåòêå. Òðàíñëîêàöèÿ Drosha â öèòîïëàçìóèíôèöèðîâàííûõ ÐÍÊ-âèðóñàìè êëåòîê è öèòîïëàçìàòè-÷åñêàÿ ðèáîíóêëåàçíàÿ àêòèâíîñòü äàííîãî ôåðìåíòà âîòíîøåíèè âèðóñíûõ äöÐÍÊ, ïðèâîäÿùàÿ ê èõ äåãðàäà-öèè, áûëà âïîñëåäñòâèè îõàðàêòåðèçîâàíà êàê ðàíåå íå-èçâåñòíûé ñïîñîá àíòèâèðóñíîé çàùèòû êëåòîê ìëåêîïè-òàþùèõ, íåçàâèñèìûé êàê îò àíòèâèðóñíîé êèÐÍÊ-èí-òåðôåðåíöèè, òàê è îò èíòåðôåðîíîâîãî îòâåòà (Shapiroet al., 2014).

Òàêèì îáðàçîì, ïðåïÿòñòâèÿ ê êîäèðîâàíèþ ìèê-ðîÐÍÊ â ãåíîìàõ ÐÍÊ-ñîäåðæàùèõ âèðóñîâ è èõ ýêñïðåñ-ñèè â èíôèöèðîâàííûõ êëåòêàõ íå ÿâëÿþòñÿ ôóíäàìåí-òàëüíî íåïðåîäîëèìûìè. Â ïîñëåäíèå ãîäû â íåñêîëüêèõïóáëèêàöèÿõ ïðèâîäÿòñÿ äîêàçàòåëüñòâà ñóùåñòâîâàíèÿìèêðîÐÍÊ (èëè «ìèêðîÐÍÊ-ïîäîáíûõ ìîëåêóë») â ãåíî-ìàõ íåñêîëüêèõ ÐÍÊ-âèðóñîâ — âèðóñà Çàïàäíîãî Íèëà(Flaviviridae) (Hussain et al., 2012), âèðóñà ãåïàòèòà À (Pi-cornaviridae) (Shi et al., 2014) è âèðóñà Ýáîëà (Filoviridae)(Liang et al., 2014). Ýòè âàæíûå ðåçóëüòàòû íóæäàþòñÿ âäàëüíåéøåé âåðèôèêàöèè, îäíàêî, ïî-âèäèìîìó, ìèê-ðîÐÍÊ-èíòåðôåðåíöèÿ íà îñíîâå âèðóñíûõ ìèêðîÐÍÊ íåîãðàíè÷èâàåòñÿ ãðóïïàìè ÄÍÊ-âèðóñîâ è ðåòðîâèðóñîâ(Swaminathan et al., 2013).

Âçàèìîäåéñòâèÿ âèðóñàñ ìèêðîÐÍÊ êëåòêè-õîçÿèíà:

îáùèå ìåõàíèçìû

Â èíôèöèðîâàííîé êëåòêå âèðóñ âñòóïàåò âî âçàèìî-äåéñòâèÿ ñ ñèñòåìîé êëåòî÷íûõ ìèêðîÐÍÊ. ×àñòü èç ýòèõâçàèìîäåéñòâèé ïðåäïîëîæèòåëüíî ìîæíî îõàðàêòåðèçî-âàòü êàê àíòèâèðóñíûå, ÿâëÿþùèåñÿ êîìïîíåíòîì ïðîòè-âîâèðóñíîé çàùèòû êëåòêè-õîçÿèíà, à äðóãóþ ÷àñòü —êàê ïðîâèðóñíûå, ÿâëÿþùèåñÿ ÷àñòüþ ðåïëèêàòèâíîéñòðàòåãèè âèðóñà.

Â öåëîì ðÿäå ðàáîò ìÐÍÊ âèðóñîâ áûëè îïðåäåëåíûâ êà÷åñòâå ìèøåíåé äëÿ ìíîæåñòâà ìèêðîÐÍÊ èíôèöè-ðîâàííîé êëåòêè (â áîëüøèíñòâå ñëó÷àåâ íà îñíîâåêîìïüþòåðíûõ ïðåäñêàçàíèé è èñïîëüçîâàíèÿ ðåïîðòåð-íûõ ñèñòåì) (Skalsky, Cullen, 2010; Ojha et al., 2016).

Âûñêàçûâàëèñü ïðåäïîëîæåíèÿ, ÷òî âçàèìîäåéñòâèÿêëåòî÷íûõ ìèêðîÐÍÊ è âèðóñíûõ ìÐÍÊ íîñÿò àíòèâè-ðóñíûé õàðàêòåð, òàê êàê îíè â ïðèíöèïå ñïîñîáíû ïî-äàâëÿòü ýêñïðåññèþ âèðóñíûõ ãåíîâ (Watanabe et al.,2007). Â íåêîòîðûõ ñëó÷àÿõ àíòèâèðóñíûé ýôôåêò áûëïðîäåìîíñòðèðîâàí äëÿ êëåòîê, îâåðýêñïðåññèðóþùèõäàííûå ìèêðîÐÍÊ (Russo, Potenza, 2011). Îäíàêî èñòèí-íóþ ñòåïåíü áèîëîãè÷åñêîé çíà÷èìîñòè òàêèõ âçàèìî-äåéñòâèé â áîðüáå îðãàíèçìà ñ âèðóñàìè îöåíèòü òðóäíî.Ñèñòåìà ìèêðîÐÍÊ ÿâëÿåòñÿ êîíñåðâàòèâíîé, ìíîãèåìèêðîÐÍÊ â íåèçìåííîì âèäå ýêñïðåññèðóþòñÿ â ðàçíûõãðóïïàõ îðãàíèçìîâ, ýâîëþöèîííî äàëåêî îòñòîÿùèõäðóã îò äðóãà. Â òî æå âðåìÿ îáùåèçâåñòíî, ÷òî âèðóñû,âî-ïåðâûõ, îáû÷íî èìåþò óçêèé ðåïåðòóàð õîçÿåâ è,âî-âòîðûõ, áûñòðî ýâîëþöèîíèðóþò. Â ýòîé ñâÿçè íà-ïðàâëåííàÿ ýâîëþöèÿ êëåòî÷íûõ ìèêðîÐÍÊ íà óçíàâàíèåâèðóñíûõ ìÐÍÊ-ìèøåíåé òðóäíî ïðåäñòàâèìà. Ñêîðåå,íàïðîòèâ, ìíîãèå âèðóñû àêòèâíî ýâîëþöèîíèðóþò âñòîðîíó èçáåãàíèÿ óçíàâàíèÿ ñâîèõ ìÐÍÊ õîçÿéñêèìèRISC-êîìïëåêñàìè. Íàïðàâëåíèÿìè òàêîé ýâîëþöèèïðåäïîëîæèòåëüíî ÿâëÿþòñÿ óêîðî÷åíèå 3R-UTR âèðóñ-íûõ ìÐÍÊ â ðÿäå ñåìåéñòâ âèðóñîâ, à òàêæå ñóùåñòâåí-íîå óñëîæíåíèå âòîðè÷íîé ñòðóêòóðû 3R-UTR ìíîãèõ âè-ðóñíûõ ìÐÍÊ, ÷òî çàòðóäíÿåò ñâÿçûâàíèå ñ íèìè êîì-ïëåêñîâ RISC (Cullen, 2013).

Êðîìå òîãî, áîëüøèíñòâî ìèêðîÐÍÊ â êëåòêå ïðèñóò-ñòâóåò â ìàëîì ÷èñëå êîïèé è ïîýòîìó èìååò ñëèøêîìíèçêèé ïîòåíöèàë ñàéëåíñèíãà äëÿ ñêîëüêî-íèáóäü çà-ìåòíîãî ñíèæåíèÿ ìîùíîãî óðîâíÿ ýêñïðåññèè âèðóñíûõãåíîâ (tenOever, 2013).

Â óæå óïîìèíàâøåìñÿ èññëåäîâàíèè (Bogerd et al.,2014) áûëî óñòàíîâëåíî, ÷òî â êëåòêàõ ñ îòñóòñòâóþùåéàêòèâíîñòüþ ãåíà Dicer ðÿä âèðóñîâ ðåïëèöèðóåòñÿ ñ òîéæå ýôôåêòèâíîñòüþ, ÷òî è â èíòàêòíûõ êëåòêàõ. Ýòè ðå-çóëüòàòû ñòàâÿò ïîä ñîìíåíèå íå òîëüêî ðîëü êèÐÍÊ âè-ðóñíîãî ïðîèñõîæäåíèÿ (î ÷åì óæå ãîâîðèëîñü ðàíåå), íîè ðîëü êëåòî÷íûõ ìèêðîÐÍÊ (òàêæå ÿâëÿþùèõñÿ ïðîäóê-òîì Dicer) â ïðîòèâîâèðóñíîé çàùèòå êëåòîê ìëåêîïèòà-þùèõ.

Áîëåå òîãî, â íåêîòîðûõ ñëó÷àÿõ ñâÿçûâàíèå êëåòî÷-íûõ ìèêðîÐÍÊ ñ âèðóñíûìè ÐÍÊ, íàïðîòèâ, íîñèò ÿâíîïðîâèðóñíûé õàðàêòåð. Íàèáîëåå èññëåäîâàííûì ïðèìå-ðîì ïîäîáíîãî ðîäà ÿâëÿåòñÿ miR-122, îáèëüíî ýêñïðåñ-ñèðóåìàÿ â ïå÷åíè. miR-122 èìååò 2 àòèïè÷íûõ ñàéòàñâÿçûâàíèÿ ñ 5R-íåêîäèðóþùåé îáëàñòüþ ãåíîìíîé ÐÍÊâèðóñà ãåïàòèòà Ñ (ÂÃÑ) (Henke et al., 2008). Ïðè èíôåê-öèè miR-122 ôîðìèðóåò îëèãîìåðíûé êîìïëåêñ ñ ÐÍÊÂÃÑ (Machlin et al., 2011), òåì ñàìûì çàùèùàÿ åå íåêýïè-ðîâàííûé 5R-êîíåö îò âîçäåéñòâèÿ êëåòî÷íûõ ýêçîíóê-ëåàç. Òàêèì îáðàçîì, miR-122 ÿâëÿåòñÿ âàæíåéøèì ôàê-òîðîì ïàòîãåííîñòè ÂÃÑ, èãðàþùèì êëþ÷åâóþ ðîëü âðåïëèêàöèè è äëèòåëüíîì ïåðñèñòèðîâàíèè äàííîãî âè-ðóñà. Áëîêèðîâàíèå ôóíêöèè miR-122 â èíôèöèðîâàííûõãåïàòîöèòàõ îêàçàëîñü ìíîãîîáåùàþùåé òåðàïåâòè÷å-ñêîé ñòðàòåãèåé â áîðüáå ñ ÂÃÑ (Lanford et al., 2010).

Îäíîâðåìåííî ÐÍÊ ÂÃÑ ñëóæèò ìîëåêóëÿðíîé ãóá-êîé, ñåêâåñòèðóþùåé miR-122 è ñîîòâåòñòâåííî äåðå-ïðåññèðóþùåé åå êëåòî÷íûå ìèøåíè (Luna et al., 2010),ñðåäè êîòîðûõ, â ÷àñòíîñòè, ðÿä äåòåðìèíàíò ìåòàñòàçè-ðîâàíèÿ ãåïàòîöåëëþëÿðíîé êàðöèíîìû (Tsai et al.,2009).

Ôóíêöèîíèðîâàíèå âèðóñíûõ ÐÍÊ â êà÷åñòâå ìîëå-êóëÿðíûõ ãóáîê, àäñîðáèðóþùèõ íà ñâîè ñàéòû ñâÿçû-âàíèÿ êëåòî÷íûå ìèêðîÐÍÊ (ïî-âèäèìîìó, ñ èõ ïîñëå-äóþùåé äåãðàäàöèåé) è òåì ñàìûì up-ðåãóëèðóþùèõ


ýêñïðåññèþ îïðåäåëåííûõ êëåòî÷íûõ ãåíîâ â öåëÿõ ïðî-ëèôåðàöèè èëè ïåðñèñòèðîâàíèÿ âèðóñà, î÷åâèäíî, ÿâëÿ-åòñÿ äîñòàòî÷íî ðàñïðîñòðàíåííûì ÿâëåíèåì, ìíîãîêðàò-íî âîçíèêàâøèì â ýâîëþöèè âèðóñîâ (McCaskill et al.,2015). Ïîìèìî âûøåóïîìÿíóòîé miR-122 è ÐÍÊ ÂÃÑ ïî-äîáíûå ôóíêöèîíàëüíûå âçàèìîîòíîøåíèÿ ïðîäåìîíñò-ðèðîâàíû äëÿ ðÿäà êëåòî÷íûõ ìèêðîÐÍÊ è íåñêîëüêèõãåðïåñ-âèðóñîâ (Guo, Steitz, 2014).

Â íåñêîëüêèõ íåäàâíèõ ïóáëèêàöèÿõ áûëà ïðåäëîæå-íà ãèïîòåçà «êîíêóðèðóþùèõ ýíäîãåííûõ ÐÍÊ» (Tayet al., 2014) èëè ïðèìåíèòèòåëüíî ê âèðóñàì «êîíêóðèðó-þùèõ âèðóñíûõ è õîçÿéñêèõ ÐÍÊ» (Li et al., 2014). Àâòî-ðû ïðåäïîëàãàþò, ÷òî âèðóñíûå ìÐÍÊ è êëåòî÷íûåìÐÍÊ â èíôèöèðîâàííîé êëåòêå, èìåþùèå ñàéòû óçíà-âàíèÿ îäíèõ è òåõ æå ìèêðîÐÍÊ, âñòóïàþò â êîíêóðåíòíûåîòíîøåíèÿ çà èõ ñâÿçûâàíèå. Ýòè âçàèìîäåéñòâèÿ ïðèâîäÿòê ðåöèïðîêíîé ðåãóëÿöèè â ñèñòåìå âèðóñíàÿ ÐÍÊ—êëå-òî÷íàÿ ìèêðîÐÍÊ—êëåòî÷íàÿ ìÐÍÊ è â êîíå÷íîì èòîãåñîçäàþò îïòèìàëüíûå óñëîâèÿ äëÿ ðåïëèêàöèè è (èëè)ïåðñèñòèðîâàíèÿ âèðóñà. Òàêèì îáðàçîì, âîçìîæíî, èìåí-íî â êîíêóðåíòíîì ñâÿçûâàíèè êëåòî÷íûõ ìèêðîÐÍÊ èäåðåïðåññèè èõ êëåòî÷íûõ ìèøåíåé, à íå â ïðÿìîì àíòè-âèðóñíîì äåéñòâèè è ñîñòîèò áèîëîãè÷åñêàÿ ðîëü áîëü-øèíñòâà âûÿâëåííûõ èëè ïðåäñêàçàííûõ âçàèìîäåéñòâèéâèðóñíûõ ÐÍÊ è ìèêðîÐÍÊ èíôèöèðîâàííîé êëåòêè.

Åùå îäíîé ñòîðîíîé ïðîáëåìû âçàèìîäåéñòâèÿ âè-ðóñîâ ñ ìèêðîÐÍÊ èíôèöèðîâàííîé êëåòêè ÿâëÿåòñÿ èç-ìåíåíèå ïðîôèëÿ êëåòî÷íûõ ìèêðîÐÍÊ, íå ñâÿçàííîãîíàïðÿìóþ ñ óçíàâàíèåì èìè âèðóñíûõ ÐÍÊ. Îöåíêà äè-íàìèêè êëåòî÷íûõ ìèêðîÐÍÊ â óñëîâèÿõ ðàçëè÷íûõ âè-ðóñíûõ èíôåêöèé ïðîâîäèëàñü íåîäíîêðàòíî (Guo, Steitz,2014). Íàèáîëåå ÿðêèì ïðèìåðîì ïîäîáûõ èçìåíåíèéïðîâèðóñíîãî õàðàêòåðà ÿâëÿåòñÿ 1000-êðàòíûé ðîñò êîí-öåíòðàöèè miR-155 â B-ëèìôîöèòàõ ÷åëîâåêà, ëàòåíòíîèíôèöèðîâàííûõ âèðóñîì Ýïøòåéíà—Áàððà (Yin et al.,2008). Ìèøåíè miR-155 âîâëå÷åíû â BACH-1-îïîñðåäî-âàííóþ ðåãóëÿöèþ òðàíñêðèïöèè (Yin et al., 2008), â ñèã-íàëüíûé ïóòü NF-kB (Lu et al., 2008) è â ðåãóëÿöèþ ïðî-ëèôåðàöèè B-êëåòîê (Loeb et al., 2012).

Èçìåíåíèå óðîâíÿ ýêñïðåññèè ìèêðîÐÍÊ ìîæåòáûòü èñïîëüçîâàíî è êëåòêîé-õîçÿèíîì â öåëÿõ áîðüáûñ âèðóñîì. Â ÷àñòíîñòè, ñóùåñòâóåò òåñíåéøàÿ ñâÿçüìåæäó àíòèâèðóñíûì èíòåðôåðîíîâûì îòâåòîì è ìèê-ðîÐÍÊ-ðåãóëÿöèåé êëåòî÷íûõ ãåíîâ. Òàê, îäíèì èçèíòåðôåðîíñòèìóëèðóåìûõ ãåíîâ ÿâëÿåòñÿ CH25H. Õîëå-ñòåðèí-25-ãèäðîêñèëàçà êàòàëèçèðóåò ïðåâðàùåíèå õîëå-ñòåðèíà â àíòèâèðóñíûé ëèïèäíûé àãåíò 25-ãèäðîêñèõî-ëåñòåðîë (25-HC) (Liu et al., 2013). 25-HC èìååò íåñêîëü-êî ýôôåêòîðíûõ àíòèâèðóñíûõ ìåõàíèçìîâ, îäíèì èçêîòîðûõ ÿâëÿåòñÿ up-ðåãóëÿöèÿ miR-130b è miR-185. Ìè-øåíÿìè ýòèõ ìèêðîÐÍÊ ÿâëÿþòñÿ ãåíû ëèïèäíîãî ìåòà-áîëèçìà, â ÷àñòíîñòè AGPAT3, SREBP2, SCARB1, LDLR,FADS1 è PPARã. Ïîäàâëåíèå èõ ýêñïðåññèè, ïî-âèäèìî-ìó, îïðåäåëÿåò àíòèâèðóñíûé ýôôåêò miR-185 â îòíîøå-íèè ðÿäà âèðóñîâ (Singaravelu et al., 2015).

Äðóãèì ïðèìåðîì ïîäîáíîãî ðîäà ÿâëÿåòñÿ up-ðåãó-ëÿöèÿ miR-342-5p èíòåðôåðîíèíäóöèðóåìûì òðàíñêðèï-öèîííûì ôàêòîðîì IRF1. Ñðåäè ìíîãî÷èñëåííûõ ìèøå-íåé miR-342-5p îáíàðóæåíû ôåðìåíòû IDI1 è SC4MOL,âîâëå÷åííûå â áèîñèíòåç ñòåðîëîâ. Èõ ðåïðåññèÿ â êî-íå÷íîì èòîãå ïðèâîäèò ê ñíèæåíèþ óðîâíÿ õîëåñòåðèíàâ èíôèöèðîâàííîé êëåòêå è, ïî-âèäèìîìó, îïîñðåäóåòïðîòèâîâèðóñíûé ýôôåêò miR-342-5p â îòíîøåíèè ÂÃÀ(H1N1), âèðóñà ïðîñòîãî ãåðïåñà 1, öèòîìåãàëîâèðóñà ÷å-ëîâåêà è ìûøè (Robertson et al., 2016).

Äèíàìèêà êëåòî÷íûõ ìèêðîÐÍÊïðè èíôåêöèè ÂÃÀ

Â ðÿäå ðàáîò óñòàíîâëåíî, ÷òî ÂÃÀ, êàê è äðóãèå âè-ðóñû, èíèöèèðóåò èçìåíåíèå ïðîôèëÿ êëåòî÷íûõ ìèê-ðîÐÍÊ. Îñíîâíûìè ìåòîäàìè ïðîôèëèðîâàíèÿ ìèê-ðîÐÍÊ ÿâëÿþòñÿ ñêðèíèíã ñ ïîìîùüþ ìèêðî÷èïîâ (mic-roarray analysis) è ñåêâåíèðîâàíèå íîâîãî ïîêîëåíèÿ. Äëÿâàëèäàöèè ïîëó÷àåìûõ ðåçóëüòàòîâ îáû÷íî èñïîëüçóþò-ñÿ êîëè÷åñòâåííàÿ ÏÖÐ â ðåàëüíîì âðåìåíè è ëþöèôå-ðàçíàÿ ðåïîðòåðíàÿ ñèñòåìà. Â íåäàâíèõ èíòåãðàòèâíûõèññëåäîâàíèÿõ ðå÷ü èäåò î ñîòíÿõ ìèêðîÐÍÊ, óðîâåíü êî-òîðûõ çíà÷èìî èçìåíÿåòñÿ ïðè âèðóñíîé èíôåêöèè (Tanet al., 2014; Bao et al., 2015; Peng et al., 2015; Makkochet al., 2016). Íà íàø âçãëÿä, íàèáîëåå ÿñíûå â ñìûñëå âî-âëå÷åííûõ ìèøåíåé è âíóòðèêëåòî÷íûõ ïðîöåññîâ ðå-çóëüòàòû èññëåäîâàíèé äèíàìèêè êëåòî÷íûõ ìèêðîÐÍÊïðè ãðèïïîçíîé èíôåêöèè ñóììèðîâàíû â òàáë. 2.

Èç ïðèâåäåííûõ â òàáë. 2 äàííûõ î÷åâèäíî, ÷òî èí-ôåêöèÿ ÂÃÀ èçìåíÿåò óðîâåíü ìíîæåñòâà ìèêðîÐÍÊ, âî-âëå÷åííûõ â âàæíåéøèå êëåòî÷íûå ïðîöåññû. Îäíèì èçíèõ ÿâëÿåòñÿ èíäóêöèÿ àïîïòîçà èíôèöèðîâàííûõ êëåòîê ïîíåñêîëüêèì âîâëåêàþùèì ìèêðîÐÍÊ ìåõàíèçìàì. Àïîï-òîç èíôèöèðîâàííûõ êëåòîê ìîæíî ðàññìàòðèâàòü êàêñðåäñòâî ïðîòèâîâèðóñíîé çàùèòû îðãàíèçìà, íàïðàâëåí-íîå íà îãðàíè÷åíèå ðåïëèêàöèè âèðóñà. Îäíàêî îäíîâðå-ìåííî àïîïòîç âíîñèò âêëàä â òÿæåñòü çàáîëåâàíèÿ. Ìàñ-ñîâàÿ àïîïòîòè÷åñêàÿ ãèáåëü ëåãî÷íûõ ýïèòåëèàëüíûõêëåòîê ñòàíîâèòñÿ îäíèì èç çâåíüåâ ôàòàëüíîãî îñòðîãîðåñïèðàòîðíîãî äèñòðåññ-ñèíäðîìà (Short et al., 2014).

Çíà÷èòåëüíîå êîëè÷åñòâî ìèêðîÐÍÊ âîâëå÷åíî â çà-âèñèìóþ îò ÂÃÀ ìîäóëÿöèþ âðîæäåííîãî èììóíèòåòàïî íåñêîëüêèì ïóòÿì (òàáë. 2). Â ÷àñòíîñòè, up-ðåãóëÿöèÿðÿäà ìèêðîÐÍÊ (miR-21, miR-451 è miR-141) ïðè èíôåê-öèè ÂÃÀ ïîäàâëÿåò ýêñïðåññèþ ïðîâîñïàëèòåëüíûõ öè-òîêèíîâ. Ñ îäíîé ñòîðîíû, èíãèáèðîâàíèå ïðîâîñïàëè-òåëüíûõ öèòîêèíîâ ìîæåò îêàçàòüñÿ âûãîäíûì âèðóñó.Âìåñòå ñ òåì èçáûòî÷íîå íåêîíòðîëèðóåìîå îñâîáîæäå-íèå öèòîêèíîâ (öèòîêèíîâûé øòîðì) ÿâëÿåòñÿ îäíèì èçíàèáîëåå îïàñíûõ äëÿ æèçíè ïîñëåäñòâèé ãðèïïà (Teija-ro, 2015), ïîýòîìó îïîñðåäîâàííûé ìèêðîÐÍÊ ÏÒÃÑïðîâîñïàëèòåëüíûõ öèòîêèíîâ, ïî-âèäèìîìó, ÿâëÿåòñÿäîïîëíèòåëüíûì ìåõàíèçìîì êëåòî÷íîãî êîíòðîëÿ âîñ-ïàëåíèÿ, ñâÿçàííîãî ñ èíôåêöèåé ÂÃÀ.

Ê ÷èñëó ñîáûòèé, ñîïðîâîæäàþùèõ ïîçäíèå ñòàäèèèíôåêöèè ÂÃÀ, îòíîñÿòñÿ èçìåíåíèÿ ïðîôèëÿ ìèê-ðîÐÍÊ, ìèøåíè êîòîðûõ âîâëå÷åíû â êëåòî÷íóþ ïðîëè-ôåðàöèþ è ðåïàðàöèþ ÄÍÊ, ÷òî, î÷åâèäíî, ñâÿçàíî ñ ðå-ãåíåðàòèâíûìè ïðîöåññàìè â ëåãî÷íîì ýïèòåëèè.

Äèíàìèêà íåêîòîðûõ êëåòî÷íûõ ìèêðîÐÍÊ áûëàîõàðàêòåðèçîâàíà êàê âûãîäíàÿ âèðóñó. Òàê, íàáëþäàå-ìîå ñíèæåíèå êîíöåíòðàöèè miR-24 ïðèâîäèò ê up-ðåãó-ëÿöèè ïðîòåàçû ôóðèíà, ïðîòåîëèòè÷åñêàÿ àêòèâíîñòüêîòîðîé â îòíîøåíèè ïðåäøåñòâåííèêà ãåìàããëþòèíèíàH0 íåîáõîäèìà äëÿ ïðîäóêöèè èíôåêöèîííûõ âèðèîíîâH5N1, ñïîñîáíûõ ê ìåìáðàííîìó ñëèÿíèþ (Lovedayet al., 2015).

Ñõîäíûì îáðàçîì down-ðåãóëÿöèÿ miR-17-3p èmiR-221 ïðè èíôåêöèè ÂÃÀ ïðèâîäèò ê äåðåïðåññèè èõìèøåíè GalNAc transferase 3 (GALNT3), êîòîðàÿ êàòà-ëèçèðóåò Î-ãëèêîçèëèðîâàíèå ìóöèíîâîãî òèïà â èíôè-öèðîâàííûõ êëåòêàõ. O-ãëèêîçèëèðîâàíèå êëåòî÷íûõðåöåïòîðîâ ÿâëÿåòñÿ îäíèì èç êëþ÷åâûõ ôàêòîðîâ, îïðå-äåëÿþùèõ àäñîðáöèþ ÂÃÀ âèðèîíîâ íà êëåòî÷íîé ïî-âåðõíîñòè (Nakamura et al., 2015).




Èçìåíåíèå ýêñïðåññèè êëåòî÷íûõ ìèêðîÐÍÊ, èõ ìèøåíè è êëåòî÷íûå ïðîöåññû,â êîòîðûå îíè âîâëå÷åíû ïðè èíôèöèðîâàíèè ÂÃÀ

ìèêðîÐÍÊÄèíàìèêàìèêðîÐÍÊ

ÌèøåíüìèêðîÐÍÊ

Ôóíêöèÿ áåëêàÏîäòèïâèðóñà


À ï î ï ò î ç

miR-29c � BCL2L2 Àíòèàïîïòîòè÷åñêàÿ ìîäóëÿöèÿ êëåòî÷íîéãèáåëè

H1N1 Guan et al., 2012

Ñåìåéñòâî miR-30 � STK17b Ïðîàïîïòîòè÷åñêàÿ êèíàçà H5N1 Li et al., 2011

miR-34a � Bax Òðèããåð àïîïòîòè÷åñêîé ïåðìåàáèëèçàöèèìåìáðàíû ìèòîõîíäðèé

H1N1 Fan, Wang, 2016

miR-34c � SIRT1 Àïîïòîòè÷åñêàÿ ìîäóëÿöèÿ äåàöåòåëèðîâà-íèÿ

» Rivera et al., 2016

miR-223 � HOXC6 Àíòèàïîïòîòè÷åñêèé òðàíñêðèïöèîííûéôàêòîð

H5N1 Li et al., 2011

� PI3K, PP2A,PKA

Îïîñðåäóþò àíòèàïîïòîòè÷åñêèé êëåòî÷-íûé ïóòü CREB

H1N1 Li et al., 2010

miR-548an � NS1ABP Àíòèàïîïòîòè÷åñêàÿ ìîäóëÿöèÿ êëåòî÷íîéãèáåëè

» Othumpangat et al.,2013

miR-4276 � COX6C Èíäóêöèÿ êàñïàçû 9 » Othumpangat et al.,2014

È ì ì ó í í û é î ò â å ò

miR-16 � TNF-a Ïðîâîñïàëèòåëüíûå öèòîêèíû H5N3 Wang et al., 2009b

miR-21 � IL1B, IL12 H1N2 Skovgaard et al., 2013

� H1N1 Li et al., 2010

miR-141 � TGF-b2 H5N1 Lam et al., 2013

miR-451 � IL6 H1N2 Skovgaard et al., 2013

miR-15b-3p � IKBKB, TRAF6,IRAK1, TRAF3

Âîâëå÷åíû â êëåòî÷íûå ïóòè Toll-ïîäîá-íûõ ðåöåïòîðîâ

H1N1 Huang et al., 2015

miR-29 � DNMT3a,DNMT3b

Ýïèãåíåòè÷åñêèå ñóïðåññîðû äëÿ COX2 èñèíòåçà IFN-l1

H1N1,H3N2

Fang et al., 2012

miR-124-3p � TRAF2, MAP3K1,MAP3K7IP1,

MAP3K7IP3, JAK1,STAT6, STAT5B

Âîâëå÷åíû â êëåòî÷íûå ñèãíàëèíãîâûåïóòè âðîæäåííîãî èììóíèòåòà

H1N1 Huang et al., 2015

miR-132 � MAPK3 Êëþ÷åâîé êîìïîíåíò êàñêàäà MAPK/ERK H1N1,H3N2

Buggele et al., 2012

miR-146a � IRAK-1 Ìåäèàòîð ñèãíàëèíãà èíòåðëåéêèíà-1 H1N1,H3N2

Buggele et al., 2012;Terrier et al., 2013

miR-200a � IFNAR1, STAT2 Âîâëå÷åíû â ñèñòåìó ñèãíàëèíãà IFN- I H1N1 Li et al., 2010

miR-449b � HDAC1 Ñóïðåññîð IFNb-ïðîìîòîðà H1N1,H3N2

Buggele et al., 2013

miR-650 � MxA Àíòèâèðóñíûé áåëîê, ñòèìóëèðóåìûé IFN H1N1 Pichulik et al., 2016

miR-664 � LIF Âîâëå÷åí â ERK5 ñèãíàëüíóþ ñèñòåìó H7N9 Wolf et al., 2016

Ê ë å ò î ÷ í à ÿ ï ð î ë è ô å ð à ö è ÿ

miR-21 � hMSH2 Ó÷àñòâóåò â ðåãóëÿöèè êëåòî÷íîãî öèêëàêëåòîê ëåãî÷íîãî ýïèòåëèÿ

H1N1 Tan et al., 2014

miR-28-5p � BRCA1 Ó÷àñòâóåò â ñèñòåìå ðåïàðàöèè ÄÍÊ » Liu et al., 2014

miR-29a, miR-29b � Akt3 Ó÷àñòâóåò â ðåãóëÿöèè êëåòî÷íîãî öèêëà » Liu et al., 2010

miR-141 � EGFR Ðåãóëèðóåò ïðîëèôåðàöèþ êëåòîê ëåãî÷íî-ãî ýïèòåëèÿ

H1N1,H5N1

Vela et al., 2014; Liet al., 2015b

Ï ð î â è ð ó ñ í î å â ë è ÿ í è å í à ý ê ñ ï ð å ñ ñ è þ ã å í î â ê ë å ò ê è - õ î ç ÿ è í à

miR-9 � MCPIP1 Äåãðàäàöèÿ âèðóñíîé ÐÍÊ H1N1,H3N2

Dong et al., 2016

miR-17-3p, miR-221 � GALNT3 Îñóùåñòâëÿåò Î-ãëèêîçèëèðîâàíèå ìóöè-íîâîãî òèïà â çàðàæåííûõ êëåòêàõ

H1N1,H3N2

Nakamura et al., 2015

Åùå îäíèì ïðèìåðîì ïðîâèðóñíîãî èçìåíåíèÿ ïðî-ôèëÿ ìèêðîÐÍÊ ïðè èíôåêöèè ÂÃÀ ÿâëÿåòñÿ up-ðåãóëÿ-öèÿ miR-9 â óñëîâèÿõ âèðóñíîãî çàðàæåíèÿ (Dong et al.,2016). Ìèøåíüþ miR-9 ÿâëÿåòñÿ áåëîê MCPIP1. Ýòîò áå-ëîê îáëàäàåò ÐÍÊàçíîé àêòèâíîñòüþ â îòíîøåíèè âèðóñ-íûõ ÐÍÊ (Lin et al., 2013). Îïîñðåäîâàííûé miR-9 ñàé-ëåíñèíã MCPIP1 ïðèâîäèò ê ñóùåñòâåííîìó óñèëåíèþðåïðîäóêöèè ÂÃÀ. Â ðÿäå ïóáëèêàöèé áûëè óñòàíîâëåíûâèðóñíûå ìÐÍÊ (ÂÃÀ) â êà÷åñòâå ìèøåíåé äëÿ êëåòî÷-íûõ ìèêðîÐÍÊ (òàáë. 3).

Âûøå ìû îáñóäèëè, ÷òî ïðÿìîé àíòèâèðóñíûé ýô-ôåêò âçàèìîäåéñòâèé êëåòî÷íûõ ìèêðîÐÍÊ ñ âèðóñíûìèÐÍÊ-òðàíñêðèïòàìè êàê ìèíèìóì íåî÷åâèäåí. Â ðÿäåñëó÷àåâ áîëåå óìåñòíî ãîâîðèòü î êîíêóðåíòíûõ îòíîøå-íèÿõ ìåæäó âèðóñíûìè è õîçÿéñêèìè ìÐÍÊ çà ñâÿçûâà-íèå ìèêðîÐÍÊ (Li et al., 2014). Â ÷àñòíîñòè, miR-491-5p,óçíàþùàÿ ìÐÍÊ PB1 (Song et al., 2010), îäíîâðåìåííîèìååò ñâîåé ìèøåíüþ ìÐÍÊ ìàòðèêñíîé ìåòàëîïðîòåè-íàçû MMP-9 (Yuan et al., 2013). MMP-9 ñâÿçàíà ñ ðÿäîìïàòîëîãè÷åñêèõ ïðîöåññîâ â ëåãêèõ, âêëþ÷àÿ îñòðûé ðåñ-



ìèêðîÐÍÊÄèíàìèêàìèêðîÐÍÊ

ÌèøåíüìèêðîÐÍÊ

Ôóíêöèÿ áåëêàÏîäòèïâèðóñà


miR-24 � Furin Ïðîòåîëèòè÷åñêîå ðàçðåçàíèå ïðåêóðñîðàãåìàããëþòèíèíà, íåîáõîäèìîå äëÿ îáðà-çîâàíèÿ èíôåêöèîííî àêòèâíîãî âèðèîíà

H5N1 Loveday et al., 2015

miR-155 � MX1 Âàæíàÿ ðîëü âî âçàèìîäåéñòâèè âèðóñ—õî-çÿèí ïðè èíôåêöèè

H1N1,H5N3

Wang et al., 2012

miR-485 � RIG-I Ïàòòåðíðàñïîçíàþùèé ðåöåïòîð H5N1 Ingle et al., 2015

Ï ð è ì å ÷ à í è å. Ñòðåëêà âíèç èëè ââåðõ ïîêàçûâàåò ñîîòâåòñòâåííî óâåëè÷åíèå èëè óìåíüøåíèå ýêñïðåññèè äàííîé ìèêðîÐÍÊ ïðè èíôåêöèèÂÃÀ.


Êëåòî÷íûå ìèêðîÐÍÊ, èìåþùèå ñâîèìè ìèøåíÿìè ìÐÍÊ ÂÃÀ

ìèêðîÐÍÊ ìÐÍÊ-ìèøåíü ÂÃÀ Ñóáòèï ÂÃÀËèòåðàòóðíûé

èñòî÷íèê

let-7c M1 H1N1 Ma et al., 2012

miR-323 PB1 » Song et al., 2010

miR-491

miR-654

miR-26a » » Tambyah et al., 2013

miR-576-3p PA

miR-628-3p HA

miR-136 PB2, HA » Scaria et al., 2006

miR-507

gga-miR-133c PB1, PB-F2, N40 » Kumar et al., 2014

gga-miR-146c*

gga-miR-1710

miR-485 PB1 H5N1 Ingle et al., 2015

miR-106 HA, NA, PA, PB1, PB2 H5N3 Wang et al., 2012

miR-142-3p M, NA

miR-146a HA, NA, NP, PB1, PB2

miR-153 HA, NA, PA, PB1, PB2

miR-155 HA, NA, NP, NS, PB1

miR-15a HA, MNP, NS, PB2

miR-17-3p HA, M, PA, PB1

miR-17-5p HA, M, NA, PA, PB1

miR-187 HA, NA, PB1, PB2

miR-18a HA, M, NA, PB1, PB2

miR-18b HA, M, NA, PB1, PB2

miR-19b HA, NS, PA, PB1

miR-202 HA, M, NA, NP, NS, PA, PB1, PB2

miR-206 HA, M, NP, NS, PB2

miR-20a HA, NA, PA, PB1, PB2

ïèðàòîðíûé äèñòðåññ-ñèíäðîì, õðîíè÷åñêóþ îáñòðóê-öèþ ëåãêèõ, àñòìó, èíôåêöèîííóþ ïíåâìîíèþ è èäèîïà-òè÷åñêèé ôèáðîç ëåãêèõ (Lee et al., 2013). Ïîýòîìó äåðåï-ðåññèÿ MMP-9 â ðåçóëüòàòå êîíêóðåíòíîãî ñâÿçûâàíèÿmiR-491-5p âèðóñíîé ìÐÍÊ îêàçûâàåòñÿ âàæíûì êîìïî-íåíòîì ïàòîãåííîñòè ÂÃÀ.

Àíàëîãè÷íûì îáðàçîì miR-29a èìååò ðÿä ïðåä-ñêàçàííûõ ìèøåíåé â ãåíîìå ÂÃÀ (Wang et al., 2012).Âìåñòå ñ òåì êëåòî÷íîé ìèøåíüþ miR-29a ÿâëÿåòñÿôîñôàòàçà PTEN, âîâëå÷åííàÿ â PI3K/Akt-ñèãíàëüíûéïóòü (Lin et al., 2016). Äåðåïðåññèÿ PTEN â ðåçóëüòàòå àä-ñîðáöèè miR-29a íà âèðóñíûå ìÐÍÊ ÿâëÿåòñÿ îäíèìèç âàæíûõ ôàêòîðîâ àïîïòîçà êëåòîê, èíôèöèðîâàííûõÂÃÀ.

Òåðàïåâòè÷åñêîå âîçäåéñòâèå íà ìèêðîÐÍÊ, âîâëå-÷åííûå â êëåòî÷íûå ïðîöåññû, ñâÿçàííûå ñ èíôåêöèåéÂÃÀ, ïðåäñòàâëÿåò ñîáîé íîâóþ ïðèâëåêàòåëüíóþ ñòðà-òåãèþ â áîðüáå ñ ãðèïïîì. Ðå÷ü ìîæåò èäòè î ââåäåíèèäîïîëíèòåëüíûõ êîïèé ìèêðîÐÍÊ (mimics), âîâëå÷åí-íûõ â àíòèâèðóñíóþ ðåãóëÿöèþ êëåòî÷íûõ ïðîöåññîâ(íàïðèìåð, â èíòåðôåðîíîâûé îòâåò), èëè, íàïðîòèâ, îáëîêèðîâàíèè ïðîâèðóñíûõ ìèêðîÐÍÊ ñ ïîìîùüþ ìî-äèôèöèðîâàííûõ àíòèñìûñëîâûõ îëèãîíóêëåîòèäîâ (an-timiRs), îáû÷íî èñïîëüçóåìûõ â âèäå antagomirs (àí-

òè-ìèêðîÐÍÊ, êîíúþãèðîâàííûå ñ õîëåñòåðèíîì) èëèLNA-îëèãîíóêëåîòèäíûõ ìîäèôèêàöèé (Stenvang et al.,2012). Íàöåëåííàÿ íà ìèêðîÐÍÊ òåðàïèÿ íà îñíîâå mi-mics èëè antimiRs âñòðå÷àåò òå æå çàòðóäíåíèÿ, ÷òî èêèÐÍÊ-òåðàïèÿ, ïðåæäå âñåãî ñîçäàíèå ýôôåêòèâíîãî èáåçîïàñíîãî ñðåäñòâà äîñòàâêè in vivo.

ÌèêðîÐÍÊ-èíòåðôåðåíöèÿ ÿâëÿåòñÿ íîâûì ïîäõî-äîì äëÿ ñîçäàíèÿ àòòåíóèðîâàííûõ ãðèïïîçíûõ âàêöèííîâîãî ïîêîëåíèÿ. Ñ ýòîé öåëüþ â íåñêîëüêèõ èññëåäî-âàíèÿõ áûë âûïîëíåí èíæèíèðèíã ÂÃÀ, âêëþ÷àþùèéâ ñåáÿ âñòðàèâàíèå ìèêðîÐÍÊ-óçíàþùåãî ýëåìåíòà(MRE) â ïîñëåäîâàòåëüíîñòü âèðóñíîãî ãåíà. Òàêèì îáðà-çîì, áûëè ñîçäàíû àòòåíóèðîâàííûå âèðóñû, ñîäåð-æàùèå MRE miR-93, âñòðîåííûé â ÎÐÑ ãåíà NP (Perezet al., 2009), è MRE miR-let-7b, âñòðîåííûé â ÎÐÑ ãåíàPB1 (Feng et al., 2015; Shen et al., 2015). Áîëåå ñëîæíûéäèçàéí àòòåíóèðîâàííîãî âèðóñà áûë èñïîëüçîâàí Ëè ññîàâòîðàìè (Li et al., 2015a). Â ýòîé ðàáîòå â âèðóñíûéãåí NS áûëà âñòðîåíà ýêñïðåññèîííàÿ êàññåòà miR-93,ýêñïðåññèðóþùàÿ èñêóññòâåííóþ ìèêðîÐÍÊ, óçíàþùóþNP. Âàêöèíàöèÿ ìûøåé ïåðå÷èñëåííûìè ðåêîìáè-íàíòíûìè âèðóñàìè ñóùåñòâåííî ñíèæàëà ñìåðòíîñòüæèâîòíûõ â ðåçóëüòàòå ëåòàëüíîé äîçû âèðóñà äèêîãîòèïà.



ìèêðîÐÍÊ ìÐÍÊ-ìèøåíü ÂÃÀ Ñóáòèï ÂÃÀËèòåðàòóðíûé

èñòî÷íèê

miR-20b HA, M, NA, NP, NS, PA, PB1, PB2

miR-211 HA, M, NA, NP, NS, PA, PB1, PB2

miR-223 HA, NA, PB1, PB2

miR-23b HA, M, NA, NP, PA, PB1, PB2

miR-24 HA, M, NA, NP, PA, PB1

miR-29a HA, M, NA, NP, NS, PB1, PB2

miR-29c HA, M, NA, NP, PA, PB1, PB2

miR-301 HA, NA, NP, PB1, PB2

miR-30b NA, NP, PB1, PB2

miR-32 HA, NS

miR-34a HA, NA, PA, PB1, PB2

miR-7b HA, M, NA, NP, NS, PA, PB1, PB2

miR-92 HA, M, NA, NP, PA, PB2

miR-216b NA H5N1, H3N2 Khongnomnan et al., 2015

miR-3145 PB1 H1N1, H5N1, H3N2

miR-3682 NS H1N1, H3N2

miR-4513 PA

miR-4753 PA, PB1 H1N1, H5N1

miR-5693 PA H5N1, H3N2

miR-127-3p PB1 H1N1, H3N2, H5N1 Makkoch et al., 2016

miR-128 PA

miR-136 NP, HA

miR-16 NP, M, ÐÀ, ÐÂ1, ÐÂ2, NS1

miR-222 NP, M, ÐÀ, ÐÂ1, ÐÂ2, NS1

miR-29a HA

miR-323 PB1

miR-548 PB1, NS1

miR-660 PA

miR-92a PB2

Çàêëþ÷åíèå

ÐÍÊ-èíòåðôåðåíöèÿ ïðåäñòàâëÿåò ñîáîé âàæíåéøèéìåõàíèçì ðåãóëÿöèè ìíîæåñòâà áèîëîãè÷åñêèõ ôóíêöèé,â ÷àñòíîñòè âçàèìîäåéñòâèé ÂÃÀ è èíôèöèðîâàííîéêëåòêè. Íåñìîòðÿ íà îïðåäåëåííîå îõëàæäåíèå îïòè-ìèçìà êîíöà 90-õ ãîäîâ ïðîøëîãî âåêà è íà÷àëà íîâîãî,ñâÿçàííîãî ñ ïåðñïåêòèâîé íåìåäëåííîãî âíåäðåíèÿ òåõ-íîëîãèè ÐÍÊ-èíòåðôåðåíöèè â êëèíè÷åñêóþ ïðàêòèêóëå÷åíèÿ ìíîæåñòâà çàáîëåâàíèé, áåññïîðíî, ÷òî òåðà-ïåâòè÷åñêîå èñïîëüçîâàíèå ìèêðîÐÍÊ- è êèÐÍÊ-âåòâåéÐÍÊ-èíòåðôåðåíöèè èìååò çíà÷èòåëüíûé ïîòåíöèàë âáîðüáå ñ ãðèïïîì. Äàííûé ïîäõîä ïîçâîëÿåò ñîçäàâàòüâûñîêîñïåöèôè÷íûå ëåêàðñòâåííûå ñðåäñòâà, íàïðàâëåí-íûå íà ÏÒÃÑ ñòðîãî îïðåäåëåííûõ öåëåâûõ ìÐÍÊ-òðàíñ-êðèïòîâ êàê âèðóñíîé, òàê è êëåòî÷íîé ïðèðîäû. Â íàñòî-ÿùåå âðåìÿ ïðîäîëæàþòñÿ àêòèâíûå ýêñïåðèìåíòàëüíûåóñèëèÿ ìíîæåñòâà ëàáîðàòîðèé ïî ñîçäàíèþ ýôôåêòèâ-íîãî è áåçîïàñíîãî ñðåäñòâà äîñòàâêè in vivo ìàëûõ ÐÍÊ.Êðîìå òîãî, äåðåãóëÿöèÿ ïðîôèëÿ ìèêðîÐÍÊ ïðè âèðóñ-íûõ èíôåêöèÿõ ÿâëÿåòñÿ ïîòåíöèàëüíî âàæíåéøèì äèà-ãíîñòè÷åñêèì èíñòðóìåíòîì.

Êîëëåêòèâ àâòîðîâ ïðèíîñèò áëàãîäàðíîñòü çà ïî-ìîùü â ïîäãîòîâêå îáçîðà Í. Í. Íèêîëüñêîìó (Èíñòèòóòöèòîëîãèè ÐÀÍ) è À. Á. Áîíäàðåíêî (Íàó÷íî-èññëåäîâà-òåëüñêèé èíñòèòóò ãðèïïà Ìèíèñòåðñòâà çäðàâîîõðàíå-íèÿ ÐÔ).

Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå Ðîñ-ñèéñêîãî íàó÷íîãî ôîíäà (ïðîåêò 15-15-00170: «Êëåòî÷-íûå ìèêðîÐÍÊ — íîâûå ìîëåêóëÿðíûå ìèøåíè äëÿ òå-ðàïèè òÿæåëûõ âèðóñíûõ èíôåêöèé»).

Ñ ï è ñ î ê ë è ò å ð à ò ó ð û

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Ïîñòóïèëà 1 III 2017

RNA INTERFERENCE AND INFLUENZA A VIRUS PATHOGENESIS

A. N. Gorshkov,1, 2, * A. V. Petrova,1 A. V. Vasin1, 3

1 Research Institute of Influenza Ministry of Healthcare of the Russian Federation, St.-Petersburg, 197376,2 Institute of Cytology RAS, St. Petersburg, 194064, and

3 Peter the Great Polytechnic University, Department of Molecular Biology, St. Petersburg, 195251;

* e-mail: [email protected]

RNA interference was discovered in the 90s during the studies of Caenorhabditis elegans nematode. Ini-tially RNA interference has been described as a selective degradation of the mRNA transcript, induced by pre-sence of short interfering RNA (siRNA) — short (20—25 b. p.) exogenous double-stranded RNA which iscomplementary to mRNA exon sites. Over the last two decades, the concept of RNA interference has becomeextended. It was recognized as one of the most important and universal elements of gene activity post-transcrip-tional regulation, which is widespread among prokaryotes, eukaryotes and viruses. In this review we provide abrief description of the various anti-viral and pro-viral RNA interference mechanisms, summarize RNA interfe-rence role in the influenza A virus biology, and discuss the opportunities and challenges of RNA interference asa new therapeutic strategy against influenza.

K e y w o r d s: RNA interference, influenza A virus, microRNA, siRNA, interferon response.


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