Disease relevance of NS2

• We have obtained the complete sequence of cloned full-length DNA (NS DNA) derived from influenza virus gene 8, which codes for two unique polypeptides, NS1 and NS2, and the sequence of the NS2 mRNA [1].
• Here we demonstrate an interaction between human CRM1 (hCRM1) and influenza A virus NS2 protein, which contains an NES motif in its N-terminal region [2].
• The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2/3 protease of hepatitis C virus [3].
• Phage display mapping identified core epitope regions recognized by mAbs NS2, NS5, CS6, CS8, and CS9 [4].
• Previous work had shown that both mRNAs are encoded by virion RNA segment 8, and that the sequences comprising the smaller of the two mRNAs (the NS2 mRNA) were also present on the NS1 mRNA [5].

High impact information on NS2

• The approximately 340 nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that NS1 and NS2 overlap by 70 amino acids that are translated from different reading frames [1].
• Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA [6].
• Actinomycin D chase experiments verified that the NS1 protein acted on the transport and not on the differential stability of NS2 mRNA in the nucleus as compared to the cytoplasm [6].
• Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production [7].
• Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1 [2].

Chemical compound and disease context of NS2

• Intratypic electrophoretic mobility differences in high resolution SDS-polyacrylamide gels were detected between corresponding matrix (M) proteins, nucleoproteins (NP), haemagglutinin (HA) and the non-structural polypeptides NS1 and NS2 induced in Vero cells by human influenza A viruses of the antigenic subtypes H1N1 and H3N2 [8].
• Isolates were also tested for their ability to act as bacterial hosts for the LPS-binding bacteriophages NS1 and NS2 [9].

Biological context of NS2

• These studies showed that the gene for NS2 reassorts with that of the nonstructural polypeptide NS1, which is coded by genome segment 8 [10].
• Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap [11].
• These positions of the mRNAs on the viral genome segment were confirmed in hybrid-arrested translation experiments using fragments of the cloned NS DNA to inhibit the synthesis in vitro of NS1 or NS2 polypeptides [12].
• Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome [12].
• The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product [13].

Anatomical context of NS2

• The cleavage activity of NS2/3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs [3].
• The glycosylation studies indicate that both amino and carboxyl termini of NS2 are located in the endoplasmic reticulum lumen [14].
• In this study, liver-infiltrating lymphocytes from persons with chronic HCV hepatitis were examined for evidence of HCV-specific CTL by using target cells infected with recombinant vaccinia viruses expressing the HCV core, E1, E2, and part of the NS2 proteins [15].
• After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent [16].
• The NS2 protein of human respiratory syncytial virus suppresses the cytotoxic T-cell response as a consequence of suppressing the type I interferon response [17].

Associations of NS2 with chemical compounds

• Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of in vitro-synthesized NS2/3 [3].
• Peptide mapping experiments indicated that polypeptides NS1 and NS2 do not share methionine- or leucine-containing tryptic peptides [12].
• Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation [18].
• Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2 [19].
• The glycoprotein genes E1 and E2, as well as the nonstructural protein genes NS2 and NS3, had the greatest genetic divergence after liver transplantation (the change in the heteroduplex mobility ratio [HMR] ranged from 2.5 to 7.0%) [20].

Regulatory relationships of NS2

• We have now found that the HRSV NS2 protein strongly controls IFN induction in mouse cells in vitro, validating the use of the mouse model to study the consequences of these gene deletions on host immunity [17].

Other interactions of NS2

• The NS1 and NS2 proteins of human respiratory syncytial virus (HRSV) have been shown to antagonize the type I interferon (IFN) response, an effect subject to host range constraints [17].
• Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution [19].
• Subcellular localization of the NS2 and NS3 proteins of hepatitis C virus was analyzed [21].
• Furthermore, these HSP90 inhibitors prevent NS2/3 cleavage when the protease is expressed in mammalian cells [3].
• To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus [22].

Analytical, diagnostic and therapeutic context of NS2

• A mRNA for NS2 has been separated from that of NS1 and the other viral polypeptides by centrifugation and has been translated in vitro [10].
• The physical association of NS2/3 with HSP90 is demonstrated by immunoprecipitation [3].
• In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE) [18].
• Molecular cloning by long distance reverse-transcription polymerase chain reaction (RT-PCR) revealed that the HCV genome in two patients with high viral loads in the liver had in-frame deletions of approximately 2 kb between E1 and NS2, which encode the E1-NS2 fusion protein and six other HCV proteins: core, NS3, NS4A, NS4B, NS5A, and NS5B [23].
• RT-PCR was carried out using primers derived from gene 8 which codes for the NS2 protein, followed by nested PCR, which confirmed the presence of group B rotavirus in all three specimens [24].

References