Problems of VirologyProblems of VirologyВопросы вирусологии0507-40882411-2097Central Research Institute for Epidemiology11806ArticlesСтатьиThe key role of Asp'6 in the proteolysis of the NP protein of influenza A virus by caspasesКлючевая роль Asp16 в протеолизе белка NP вируса гриппа А каспазами в инфицированных клетках15122003486NO6 (2003)№6 (2003)81409062023Copyright ©; 2003, Zhirnov О.P., Vorobyova I.V., Veselovsky E.M., Klenk H.Copyright ©; 2003, Жирнов О.П., Воробьева И.В., Веселовский Е.М., Klenk H.2003Zhirnov О.P., Vorobyova I.V., Veselovsky E.M., Klenk H.Жирнов О.П., Воробьева И.В., Веселовский Е.М., Klenk H.https://creativecommons.org/licenses/by/4.0https://virusjour.crie.ru/jour/article/view/11806

The main nucleocapsid protein NP (molecular weight - 56 kD) of human influenza A virus (IAV) was found to be subject to the N-terminal proteolysis in position Asp16 with production of aNP (molecular weight - 53 kD) in the infected cells' apoptosis. It was assumed that NP of avian and animal influenza viruses was not subject to proteolysis since it has Gly'6. To verify the above assumption the NP chimeric gene of human influenza virus was developed; Asp16 was replaced by Gly by means of "site-oriented" mutagenesis in the above gene, after that, the A/ WSN/33 (H1N1) mutant of human influenza virus with "avian" NP and with point mutation (Gly16) was developed by using the method of "reverse genetics". The "human" influenza virus with "avian" chimeric NP/Gly16 turned out to be viable but had a lower replication velocity versus its wild-nature counterpart. It is noteworthy, that the mutant virus caused the cellular apoptosis in the remote infection period the way the wild virus did; however, NP of the former was found to be resistant to cellular caspasas and was not subject to proteolysis in infected cells. The conclusion is that Asp16 in NP molecule of human IAV is involved into the regulation process of virus replication and is the key element in NP proteolysis by cellular caspasas in cells' apoptosis.

Установлено, что при апоптозе инфицированных клеток главный нуклеокапсидный белок NP (мол. масса 56 кД) вируса гриппа А человека претерпевает N-концевой протеолиз в позиции Asp16 с образованием продукта aNP (мол. масса 53 кД). Предполагалось, что белок NP вирусов гриппа птиц и животных не подвержен протеолизу, так как имеет Gly16. Для проверки этого предположения получали химерный ген NP вируса гриппа человека, в котором посредством сайтнаправленного мутагенеза Asp16 заменен на Gly, и с помощью метода "обратной генетики" получен мутант вируса гриппа человека A/WSN/33 (H1N1), несущий NP "птичьего типа" с точечной мутацией (Gly16). Обнаружено, что вирус гриппа "человека" с химерным NP/Gly16 "птичьего типа" оказался жизнеспособным, но имел сниженную скорость репродукции по сравнению с диким вирусом. При этом, подобно дикому типу, мутантный вирус вызывал апоптоз клеток на поздних сроках инфекции, однако его белок NP был резистентным к клеточным каспазам и не подвергался протеолизу в инфицированных клетках. Из этих наблюдений следует, что Asp16 в молекуле NP вируса гриппа А человека участвует в регуляции репликации вируса и играет ключевую роль в протеолизе NP клеточными каспазами при апоптозе клеток.

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