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

Abstract

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.

Novel Negative Sense Genes in the RNA Genome of Coronaviruses

“…Currently, such ambisens viruses include viruses of four genera: phlebo-, tospo-, arena-, and tenuviruses [ 15 ]. The ambisense genes located in the genome in the stacking format were found in influenza viruses, in which, similarly to coronaviruses, direct expression of these genes has not yet been identified, but there are indirect signs of such expression during natural viral infection in vivo [ 12 , 13 ]. The study of the mechanisms of the possible expression of the genetic information of these new genes, as well as the elucidation of the role and significance of the detected genes and/or their protein products during viral replication can serve as the basis for creating a new type of vaccines and antiviral chemotherapy agents for treatment of coronavirus infection.…”

“…The function and role of the newly discovered ambipolar viral genes have not yet been established. In the case of influenza viruses, there is an assumption that the identified new ambisense genes may be important in the regulation of the immune response to viral proteins and/or in the regulation of the stability of viral proteins in infected cells through the protein deubiquitination system [11][12][13][14]. To understand the possible functional significance of the identified new ambipolar genes, it is necessity to take into account two features inherent in these genes.…”

Unique Bipolar Gene Architecture in the RNA Genome of Influenza A Virus

“…Our data suggest that the variability of NSP is similar to that of the most variable glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA), located at the virion surface and representing major target mole cules for the antiviral factors of the host adaptive immu nity ( Table 2). The negative sense NS1 and NEP genes in the NS segment are less variable [17]. There are two pos sible reasons that may account for this phenomenon.…”

“…In particular, the NSP8 protein can be synthesized in the in vitro translation system by mam malian ribosomes on the full size viral RNA template [10]. Specific immune lymphocytes against this protein or its components have been found in the infected ani mals [17,22,23]. The properties of NSP8 as a "invisible protein" may be accounted by its low synthesis and/or accumulation levels, high lability, short half life, and pos sible tissue specific expression of the NSP8 gene in cer tain cell types containing factors necessary for the regula tion of the positive strand NSP gene expression.…”

“…Table 2. Evolutionary variability of proteins of the 1968 2018 influenza A virus/H3N2 * Variability ratio (VR) was calculated as the number of amino acid substitutions in the proteins from the 1968 2018 human influenza virus A/H3N2 strains per year per 100 amino acid residues in each protein [17] (GenBank data). PB1, polymerase (basic) protein 1; HA, hemagglutinin; NA, neuraminidase; M1, matrix protein 1; NS1, interferon antagonist protein 1; NEP, nuclear export protein; NSP8, negative strand protein 8.…”