The influenza C virus genome consists of seven singlestranded RNA segments of negative polarity. RNA segment 6 (M gene) of C/Yamagata/1/88 is 1,181 nucleotides in length and has a single open reading frame (positions 27 to 1148) capable of encoding a polypeptide of 374 amino acids with a predicted M r of 42,000 (9, 35). However, the predominant mRNA transcript of this RNA segment lacks nucleotides 755 to 982 and encodes a 242-amino-acid matrix (M1) protein with an M r of 27,000. Elimination of the intron results in the introduction of a termination codon (consisting of nucleotides 753, 754, and 983) after amino acid residue 242 (9, 35). Unspliced mRNA from RNA segment 6 is synthesized in infected cells, though at very low levels (13% of spliced mRNA) (9). This mRNA species is capable of coding for a 374-amino-acid protein (P42) containing an additional 132 amino acids from the C terminus of M1 (11), which is cleaved by signal peptidase to generate CM2, composed of the C-terminal 115 amino acids, in addition to the M1Ј protein, composed of the N-terminal 259 amino acids (12, 26). The CM2 protein forms a voltage-activated ion channel permeable to chloride ions (13). Recently, it has been suggested that CM2 also has pH-modulating activity (2).In influenza A virus-infected cells, the colinear transcript from segment 7 encodes M1, and the M1 mRNA also undergoes alternative splicing to generate spliced M2 mRNA and mRNA 3 . The colinear transcript and spliced mRNA from influenza A virus segment 8 are translated into NS1 and NS2 (NEP), respectively. It has been reported that the steady-state level of spliced viral transcripts is only 10% of that of unspliced viral transcripts in influenza A virus-infected cells (15,16). The inefficient splicing of viral pre-mRNAs can be understood partly by the fact that influenza A virus NS1 protein is associated with spliceosomes and inhibits pre-mRNA splicing (6,7,17). cis-acting sequences in the NS1 transcript also negatively regulates splicing (22). The splicing of influenza A virus M1 mRNA is controlled by the rate of nuclear export (34). The alternative splicing of influenza A virus M1 mRNA is regulated by the binding of the viral polymerase complex and cellular splicing factor SF2/ASF (28, 29). In the case of influenza C virus, however, the mechanism by which influenza C virus M mRNA is efficiently spliced has not yet been clarified. Influenza C virus RNA segment 7 (NS gene) encodes two nonstructural proteins, the 246-amino-acid NS1 protein (C/NS1), encoded by a colinear mRNA transcript of the gene, and the 182-amino-acid NS2 protein (C/NS2), encoded by a spliced mRNA (1,8,18) (see Fig. 5). Using transfected cells, Paragas et al. (25) have demonstrated that C/NS2 possesses nuclear export activity. We recently demonstrated that C/NS2 plays a role in the nuclear export of vRNP in influenza C virus-infected cells, and that C/NS2 is incorporated into virions, where it associates with
RNA segment 7 of influenza C virus encodes two non-structural (NS) proteins, NS1 and NS2. The influenza C virus NS2 protein has been proposed to possess nuclear export activity like that of influenza A and B virus NS2 proteins (NEP). In the present study, we investigated the kinetics and localization of the NS2 protein in influenza C virus-infected cells, and analysed whether NS2 is present in virions. Immunofluorescent staining analysis of the infected cells indicated that NS2 was localized in the nucleus immediately after synthesis and predominantly in the cytoplasm in the later stages of infection. Confocal microscopy revealed that a part of the NS2 protein was colocalized with nucleoprotein NP/vRNP in the cytoplasm and on the cell membrane in the late stages of infection. The NS2 protein was detected in influenza C virions purified by gradient centrifugations and/or affinity chromatography. Trypsin treatment demonstrated that the NS2 protein was present inside the viral envelope. Furthermore, glycerol gradient analysis of detergent-solubilized virions revealed that the NS2 protein cosedimented with vRNPs. These data suggest that the influenza C virus NS2 protein is incorporated into virions, where it associates with vRNP.
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