Cellular immune response in infected mice to NSP protein encoded by the negative strand NS RNA of influenza A virus

Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N- and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus.Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N- and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus

Клеточный иммунный ответ у инфицированных животных против белка NSP, кодируемого негативной цепью NS RNA вируса гриппа А

Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N- and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus.Вирус гриппа А относится к оболочечным RNA-содержащим вирусам с негативно-полярным геномом, состоящим из 8 сегментов RNA, каждый из которых является матрицей для синтеза позитивно-полярных mRNA, которые, в свою очередь, служат матрицей для синтеза 16 вирусных белков с использованием для некоторых механизма сплайсинга или трансляционного шифта. Восьмой сегмент NS посредством классической негативной стратегии генома кодирует белок NS1, обладающий анти-интерфероновой активностью (27 кДа), и белок ядерного экспорта NEP (NS2) (nuclear export protein, 14 кДа). Кроме этого, в сегменте NS обнаружена дополнительная открытая рамка для синтеза третьего вирусного белка по альтернативному пути посредством прямой трансляции негативно полярной вирусной RNA. Эта стратегия генома получила название позитивно-полярной. Образование данного белка, названного NSP (negative strand protein), при инфекции вируса гриппа А в целостном организме пока не обнаружено. Анализ белка NSP in silico выявил наличие доменов MHC-I (major histocompatibility complex-1) и MHC-II. Сравнительный анализ вирусов гриппа подтипа H3N2, циркулировавших среди людей в период с 1968 по 2018 гг., показал высокую изменчивость гена белка NSP, которая сходна с изменчивостью поверхностных белков гемагглютинина (HA) и нейраминидазы (NA). Наибольшая изменчивость обнаруживалась в зонах, соответствующих N- и C-концевым участкам белка NSP. В настоящей работе изучена возможность образования иммунных лейкоцитов, специфичных к белку NSP, у мышей после инфекции вирусом гриппа А. Показано, что у мышей после заражения последовательно двумя вирусами гриппа А разных сероподтипов обнаруживаются иммунные лейкоциты, специфически распознающие вирусные домены в центральной зоне белка NSP (позиции аминокислот 82-119). Полученные данные с большой вероятностью позволяют утверждать, что при инфекции вирусом гриппа А в организме животных имеет место экспрессия гена NSP, что в свою очередь подтверждает концепцию о биполярной (амбисенс) стратегии генома вируса гриппа А