into 293T cells together with four (PB2, PB1, PA, and NP) or nine (PB2, PB1, PA, HA, NP, NA, M1, M2, and NS2) viral protein expressing plasmid DNAs. D

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1 Jpn. J. Infect. Dis., 63, , 2010 Review The Molecular Virology and Reverse Genetics of Influenza C Virus Yasushi Muraki 1,2 * and Seiji Hongo 2 1 Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa ; and 2 Department of Infectious Diseases, Yamagata University Faculty of Medicine, Yamagata , Japan (Received January 18, Accepted March 29, 2010) CONTENTS 1. Introduction 2. Reverse genetics of influenza viruses 3. Molecular virology of influenza C virus 4. Epidemiology of influenza C virus 5. Generation of influenza C virus like particles and a recombinant influenza C virus 6. Analysis of the structure function relationship of the M1 protein 7. Comparison of the generation efficiencies of influenza C and A viruses 8. Prospects for research on influenza C virology 9. Conclusion SUMMARY: Influenza C virus, an enveloped virus containing seven single stranded RNA segments of negative polarity, belongs to the genus Influenza C Virus of the family Orthomyxoviridae. A number of questions remain to be resolved with regard to the molecular virology and epidemiology of the virus. To address them, we have established a virus like particle (VLP) generation system and reverse genetics of the virus and succeeded in clarifying the structure function relationship of the M1 protein of the virus. Although the approach adopted was similar to that for influenza A virus reverse genetics, the number of infectious influenza C viruses generated was much lower than that for influenza A virus. Based on a comparison of the number of influenza C VLPs with that of influenza A VLPs generated using a similar system, we proposed a virion generation mechanism unique to influenza C virus. 1. Introduction Reverse genetics, as the term is used in molecular virology, describes the generation of viruses possessing genome(s) derived from cloned cdna(s). Of the viruses belonging to the family Orthomyxoviridae, reverse genetics has hitherto been reported for influenza A, influenza B and Thogoto viruses. Recently, our research group, as well as Crescenzo Chaigne and van der Werf, reported the successful reverse genetics of influenza C virus. In this review, we will first provide an overview of research on reverse genetics of influenza A virus, and then summarize the molecular virology and epidemiology of influenza C virus, including a discussion of presently unresolved issues of the virus. In the latter part of this review, we will deal with a virus like particle (VLP) generation and reverse genetics of influenza C virus by our research group and provide a hypothesis to explain influenza C virion generation as observed in the established reverse genetics system. 2. Reverse genetics of influenzaviruses The genomes of negative sense RNA viruses, including influenza viruses, are noninfectious. Therefore, *Corresponding author: Mailing address: Department of Microbiology, Kanazawa Medical University School of Medicine, Uchinada, Ishikawa , Japan. Tel: { , Fax: { , E mail: ymuraki kanazawa med.ac.jp for the generation of negative sense RNA viruses, researchers faced the obstacle of providing the viral RNA (vrna) with viral RNA polymerase and nucleoprotein. In the case of influenza A virus, the viral ribonucleoprotein (vrnp) complex, composed of three polymerase subunits (PB2, PB1, and PA), nucleoprotein (NP) and vrna, is minimally required. Initially, by transfecting an artificially reconstituted vrnp complex into eukaryotic cells followed by infection with an influenza helper virus, successful recovery of a recombinant influenza virus containing a viral gene segment derived from the cloned cdna was reported (1,2). In 1996, Pleschka et al. reported the successful generation of a transfectant influenza virus (3). To synthesize influenza vrna in the nucleus, they made use of a nucleolar enzyme, RNA polymerase I, in a system that had been established by Zobel et al. (4). Cotransfection of the Pol Iplasmid encoding neuraminidase (NA) vrna together with viral protein expressing plasmids for PB2, PB1, PA, and NP, followed by infection with an influenza helper virus, resulted in the recovery of a recombinant containing the NA gene of interest. Even in using this method, however, the helper virus dependent system remained an obstacle to the efficient recovery of the recombinant virus. In 1999, a recombinant influenza A virus was reported to be generated entirely from cloned cdna for the first time (5). The cdnas of the eight vrna segments of A/WSN/33 or A/PR/8/34 virus were each cloned into the phh21 vector in negative sense orientation between the RNA polymerase Ipromoter and terminator sequences. The resulting eight plasmids were transfected 157

2 into 293T cells together with four (PB2, PB1, PA, and NP) or nine (PB2, PB1, PA, HA, NP, NA, M1, M2, and NS2) viral protein expressing plasmid DNAs. Despite the need for the simultaneous transfection of as many as 12 or 17 plasmids into the cells, approximately 10 6 to 10 7 infectious influenza A viruses/ml were produced at 48 to 72 h posttransfection. Thus, a recombinant influenza A virus in which all RNA segments were derived from cdna clones was generated. Improved approaches for the generation of the recombinant influenza A virus were later reported by several research groups (6 8). Furthermore, influenza B (9 11) and Thogoto viruses (12) were successfully generated in a similar way. Despite all these advances, however, no reverse genetics of influenza C virus was established. virus. Yamashita et al. cloned the three largest RNA segments of C/JJ/50 (17). A comparison of the nucleotide sequences of the segments with those of the influenza A and B viruses revealed that RNA segments 1 and 2 encode the proteins equivalent to PB2 and PB1 of the influenza A and B viruses, respectively. The protein encoded by RNA segment 3 is referred to as P3 rather than PA, since it does not display any acid charge features at a neutral ph. Evidence has been obtained to suggest that the transcription and replication of the influenza C virus genome follows the same strategy as that of influenza A virus in that the three proteins form an RNA 3. Molecular virology of influenza C virus Influenza C virus, which belongs to the genus Influenza C Virus of the family Orthomyxoviridae, was first isolated from a patient with respiratory illness in 1947 (13). It is an enveloped virus containing seven single stranded RNA segments of negative polarity and encodes the following proteins: PB2, PB1, P3, hemagglutinin esterase fusion (HEF), NP, matrix (M1) protein, and CM2, as well as the non structural proteins NS1 and NS2 (Figs. 1 and 2). The virus usually causes mild upper respiratory illness (14), but it can also cause lower respiratory infections (15,16). A number of research groups have studied the structure and function of the genes and gene products of the Fig. 1. Structure of influenza C virus. Influenza C virus has seven single stranded RNA segments of negative polarity. The viral ribonucleoprotein (vrnp) complex is composed of viral RNA (vrna) and the PB2, PB1, P3, and NP proteins. HEF forms a spike on the virion. M1 is present beneath the envelope. CM2 is the second membrane protein of the virus. A small amount of NS2 is incorporated into the virions. Fig. 2. Genome structure of influenza C/Ann Arbor/1/50 virus. RNA segments 1 (PB2 gene) to 7 (NS gene) are shown in positive sense orientation. Numbers indicate the nucleotide positions along the genes. The lines at the 5? and 3? termini represent the noncoding regions. The V shaped dotted lines in segments 6 and 7 indicate the intron. Viral proteins encoded by the genes are shown in boxes with the number of amino acids in parentheses. M1 is encoded by a spliced mrna of RNA segment 6 (M gene), into which the TGA (stop codon) is introduced as a result of splicing. The P42 protein, encoded by a collinear transcript of the M gene, is cleaved by signal peptidase at an internal cleavage site (black triangle) to generate M1' and CM2. RNA segment 7 (NS gene) encodes the NS1 and NS2 proteins. NS1 is encoded by a collinear mrna, and NS2 is encoded by a spliced mrna. The C terminal region of NS2 (gray area) contains {1 ORF. The C/Ann Arbor/1/50 strain contains a total of 12,906 nucleotides. 158

3 polymerase complex (18 20), although the precise role(s) of the respective proteins remains to be elucidated. RNA segment 4 encodes the HEF glycoprotein, which forms a spike on the virus envelope (Fig. 1). The HEF protein has three biological activities: receptor binding, receptor destroying (acetylesterase), and membrane fusing activities. The receptor of the virus is the 9 Oacetyl N acetylneuraminic acid (Neu5,9Ac 2 ), and the acetylesterase activity of HEF inactivates the virus receptor by releasing the O acetyl residues from the C 9 position of Neu5,9Ac 2 (21 23). The fusion activity of HEF is dependent on the proteolytic cleavage of a precursor (HEF 0 )intohef 1 and HEF 2, and it requires activation at a low ph upon internalization of the viral particle through the endosome pathway (24,25). Sugawara et al. produced 37 monoclonal antibodies (MAbs) against HEF and indicated the presence of nine antigenicsites(a 1toA 5andB 1toB 4)onthemolecule (26). The amino acid(s) recognized by the MAbs were identified by isolating the escape mutants of the MAbs directed against A 1 to A 4 (27). In 1998, Rosenthal et al. revealed the three dimensional structure of HEF and showed that the three biological functions were attributable to distinct domains on the molecule (28). Together with a report by Matsuzaki et al. (27), this demonstrated that the antigenic sites A 1, A 2, and A 4 were located on the receptor binding domain and A 2 on the acetylesterase domain, indicating that, like the influenza A virus hemagglutinin (HA), the main antigenic region of HEF is located on the globular head of the molecule. HEF has a number of unique characteristics that are different from those of the influenza A virus HA. There are only three amino acids in the cytoplasmic region of HEF, whereas the corresponding region of HA contains 11 to 12 amino acids. The short cytoplasmic region may or may not affect the transportation of HEF to the cell surface (29 32). The fatty acid attached to the cysteine at residue 642 of HEF is a stearic acid (33), whereas palmitic acid is mainly attached to the influenza A virus HA (34). Whether these facts are significant to the influenza C virus replication remains to be elucidated. Nakada et al. determined the entire sequence of RNA segment 5 of C/California/78 and suggested that the segment codes for NP (35). Sugawara et al. constructed a panel of MAbs against NP and reported that (i) there are at least two antigenic sites (I and II) on the molecule, (ii) the localization of NP in virus infected cells is consistent with that of the influenza A virus NP, and (iii) NP exhibits molecular maturation during transport to the nucleus in virus infected cells (36,37). NP was also shown to be essential to the transcription/replication processes of an artificial vrna flanked by the noncoding regions (NCRs) of C/Johannesburg/1/66 (19,20). Thus, although the features of the NP protein clarified to date are consistent with those of the influenza A virus NP protein, the functional domain(s) of NP remains to be analyzed. The RNA segments 6 and 7 are bicistronic genes (Fig. 2). The spliced mrna of the RNA segment 6 encodes M1 (38). M1 gives rigidity to the virion and is involved in the budding and morphogenesis processes of the virus (39 42). The unspliced mrna of the segment encodes the 374 amino acid protein, P42, which is cleaved by signal peptidase at an internal cleavage site to give M1' and CM2 (43,44). The biochemical features of CM2, the second membrane protein of the virus, are closely similar to those of M2 (45,46), a proton channel of influenza A virus. Although CM2 expressed in Xenopus laevis oocytes forms an ion channel permeable to Cl (47) and CM2 has the ability to modulate the ph of the exocytic pathway (48), the role of CM2 in the virus replication cycle remains unclear. M1', composed of the N terminal 259 amino acids of P42, is degraded shortly after cleavage through the signal located in the C terminal 17 amino acid region of the protein (49). The half life of P42 is approximately 30 min (50), although a part of P42 is transported to the cis Golgi apparatus (51). The significance of M1' and P42 in the viral life cycle also remains to be determined. Analysis of the NS gene of the C/California/78 strain initially showed that the gene contained 934 nucleotides and was capable of encoding both the 286 amino acid NS1 and 121 amino acid NS2 proteins (52,53). Based on a comparison of the nucleotide sequences from a larger number of influenza C virus isolates, including C/ California/78, the gene was later demonstrated to potentially encode NS1 (246 amino acids) from unspliced mrna and NS2 (182 amino acids) from spliced mrna (54). In fact, the 246 amino acid NS1 and 182 amino acid NS2 proteins were identified in virus infected cells (54,55). The involvement of NS1 in persistent infection (55) and viral mrna splicing (56) was also reported. Like the influenza A virus NS2/NEP, the influenza C virus NS2 possesses nuclear export activity and is incorporated into virions (57,58). Crescenzo Chaigne et al. investigated the functions of the NCR of an influenza C virus RNA segment (19,20). In their study, the model type RNA flanked by NCR of the C/Johannesburg/1/66 strain NS gene was expressed in COS 1 cells from the Pol I plasmid based system, together with the PB2, PB1, P3, and NP proteins, and the RNA template was shown to be transcribed and replicated. Thus, the involvement of the NCR of the influenza C viral genome in its replication and transcription was demonstrated. 4. Epidemiology of influenza C virus The epidemiology of influenza C virus has also been extensively investigated. In the 1980s, on the basis of the results obtained from a limited number of influenza C virus isolates, the virus was considered to be antigenically stable, divided into several antigenic groups, and evolved more slowly than influenza A virus (59 61). Moriuchi et al. developed a tissue culture method for primary virus isolation that is convenient for routine work with a large number of clinical specimens (15,62), and they then initiated surveillance for influenza C virus infection in Sendai and Yamagata Cities in Japan. The established system has enabled us to systematically analyze influenza C virus epidemiology and thereby find that (i) influenza C virus is divided into six antigenic and genetic groups (Taylor/1233/47, Aichi/1/81, Sao Paulo/378/82, Kanagawa/1/76, Yamagata/26/81, and Mississippi/80 related lineages) (Fig. 3) (63,64), (ii) the evolutionary rate of the virus is lower than that of 159

4 Fig. 3. Phylogenetic tree of influenza C virus HEF genes. The region from nucleotides 64 to 1,989 of the HEF gene was analyzed. Horizontal distances are proportional to the minimum number of nucleotide differences needed to join the sequences. Numbers above the branches are the bootstrap probabilities (z) of each branch. A total of 74 strains were divided into six lineages as indicated on the right of the figure (adapted from reference 64 with permission; kindly provided by Yoko Matsuzaki). influenza A virus (63,65); e.g., the rate of the HEF gene is 0.49 ~ 10 3 nucleotides per site per year, which is only one ninth of that of the influenza A virus HA gene, (iii) viruses belonging to different genetic and antigenic groups cocirculate within a limited geographical area during a given period, and reassortment events between different viruses frequently occur (64,66,67), and (iv) viruses containing a proper combination of genetic elements through reassortment events may have an advantage in spreading among the human population (54,64,68,69). There is evidence that influenza C virus has the potential to infect animals, as the presence of specific antibodies was demonstrated in the serum of pigs (70,71) and dogs (72 74). Furthermore, a number of influenza C viruses were isolated from abattoir pigs in Beijing, China, and pig to pig transmission of the virus on experimental infection has been demonstrated (75). Hu 160

5 man influenza C virus isolates were found to be genetically and antigenically related to pig isolates, suggesting that interspecies transmission between humans and pigs has occurred in nature (76). Ohwada et al. showed that experimental infection resulted in clinical symptoms and viral replication in dogs (77). The potential role of animals as a reservoir for human influenza C remains to be elucidated, although influenza C viruses seem to be maintained among the human population. 5. Generation of influenza C virus like particles and a recombinant influenza C virus For a number of negative sense RNA viruses, the establishment of a reverse genetics has been preceded by the construction of a mini replicon system. Therefore, we attempted to establish an influenza C VLP generation system, as the packaging of the artificial genome into influenza C virions and gene transfer to susceptible cells by VLPs had not yet been reported at that time. The cdna of the green fluorescent protein (GFP) gene flanked by the NCR sequences of RNA segment 5 (NP gene) of C/Ann Arbor/1/50 was cloned into a phh21 vector in an anti sense orientation between the Pol I promoter and the terminator sequences so that the artificial RNA (GFP vrna) was expressed under the control of the human Pol I promoter. The resulting plasmid DNA, ppoli/np AA.GFP( ), was transfected into 293T cells, a human embryonic kidney cell line constitutively expressing simian virus 40 large T antigen, together with viral protein expressing plasmids for PB2, PB1, P3, HEF, NP, M1, CM2, NS1, and NS2, and incubated for up to 72 h. Using electron microscopy, we confirmed that the supernatant of the 293T cells contained influenza C VLPs, and, by the quantification of GFP positive HMV II cells infected with the VLPs and helper virus (C/Ann Arbor/1/50), we determined that the number of VLPs generated was approximately 10 6 / ml (41). The findings by several groups suggest that influenza C virus proteins function more efficiently at 339C than at 379C. Nagele and Meier Ewert showed that the maximum RNA polymerase activity of influenza C virus was detected at 339C (18). We also observed that, in the Pol I based system, a higher amount of luciferase was expressed at 339C thanat379c (data not shown). Therefore, our experiment was carried out at 339C, not 379C, including incubation of the transfected 293T cells and infection of the HMV II cells with the VLPs (41). ``Cap snatching'' is a unique feature of the influenza A virus transcription initiation process, and the roles of the three RNA polymerase subunits involved in that process have been extensively analyzed (78 83). In our study, in determining the sequence of the 3? end of the NP gene, we found that 12 nucleotides were added to the 5? end of the NP gene mrna (unpublished results). Similar results were obtained for the other six gene segments (data not shown, see below). These findings provide evidence that the cap snatching machinery by influenza C virus polymerases is in fact activated in a similar manner as for influenza A virus, although the roles of the respective polymerase subunits in the cap snatching process remain to be clarified. In the influenza C VLP generation experiment, NS1 Fig. 4. Reverse genetics of influenza C virus. Seven Pol I plasmids for the expression of vrnas were transfected into 293T cells together with viral protein expressing plasmids for PB2, PB1,P3,HEF,NP,M1,CM2,NS1,andNS2.Recombinant influenza C viruses produced from the transfected 293T cells were then titrated and propagated in the amniotic cavity of embryonated chicken eggs. expression in the transfected 293T cells was found to be required for efficient gene transfer to HMV II cells (84). On the other hand, even without NS1 expression, influenza A VLPs were successfully generated and found capable of efficiently transmitting the synthetic reporter RNA to susceptible cells (85). This discrepancy may suggest that the influenza C virus NS1 has a unique function(s) in the virus replication cycle that differs from those reported for influenza A virus. To establish the reverse genetics of influenza C virus, we constructed Pol I plasmid DNAs for the seven RNA segments of C/Ann Arbor/1/50, a prototype strain of influenza C virus. The resulting Pol I plasmids were then transfected into 293T cells, together with four (PB2, PB1, P3, and NP) or nine (PB2, PB1, P3, HEF, NP, M1, CM2, NS1, and NS2) viral protein expressing plasmids (Fig. 4). The resultant supernatant was determined to contain 10 1 to 10 3 EID 50 /ml of infectious recombinant viruses (42), the titer of which was much lower than that of influenza A virus (10 6 to 10 7 PFU/mL) (see below). Crescenzo Chaigne and van der Werf reported the reverse genetics of influenza C virus using a similar approach by which, at 10 days posttransfection, they obtained 10 4 PFU/mL of the recombinant virus (86). This finding suggests that, as in our observation, a limited number of infectious recombinants existed in the supernatant of the transfected 293T cells. Thus helper virus 161

6 independent reverse genetics has currently become available for application to influenza C virology, thereby affording the opportunity to resolve a number of questions regarding the virus. At the moment, however, wemustadmitthatthecurrentsystemhasadisadvantage in that recombinants with severe growth defect(s) may not be obtained due to the low generation efficiency, a problem that needs to be overcome. 6. Analysis of the structure function relationship of the M1 protein Nishimura et al. reported that cord like structures (CLSs), composed of numerous filamentous particles in the process of budding, were found to extrude from C/Yamagata/1/88 infected HMV II cells (39). Each of these particles was covered with a layer of surface projections and aggregated with their long axes. Further analysis of a series of reassortant viruses between C/Yamagata/1/88 and C/Taylor/1233/47, the latter of which is a unique strain incapable of forming CLS, showed that reassortants with the M gene from C/Taylor/1233/47 could not form CLS on infected cells (40). Upon establishment of the influenza C VLP generation system, we identified CLSs extruding from the VLP generating 293T cells. By expressing a series of M1 mutants in the 293T cells together with the other plasmids required for VLP generation, we demonstrated that the M1 protein is a determinant for CLS formation and residue 24 of M1 (Ala or Thr) is responsible for CLS formation as well as VLP morphology (filamentous or spherical) (41). Furthermore, the generation of an infectious recombinant virus with an M1 mutation revealed that residue 24 of the M1 protein (Ala or Thr) also affects CLS formation on the infected cells and virion morphology (filamentous or spherical). Membrane flotation analysis of recombinant virus infected cells revealed that the wild type M1 protein (possessing Ala at residue 24) showed higher affinity to the plasma membrane than mutant M1 (possessing Thr at residue 24), suggesting that an amino acid on M1 affects the virion morphology through the membrane affinity of M1 to the plasma membrane (42). Thus, using the reverse genetics system of influenza C virus, we could demonstrate the structure function relationship of the M1 protein in the context of viral replication. 7. Comparison of the generation efficiencies of influenza C and A viruses Initially, we expected that the adoption of a similar approach would allow the recombinant influenza C virus to be generated as efficiently as influenza A virus (10 6 to 10 7 PFL/mL), since (i) the number of RNA segments in influenza C virus is seven, which is smaller by one segment than that in influenza A virus, and (ii) the number of influenza C VLPs generated using a similar system was approximately 10 6 /ml, which is 100 fold higher than that of influenza A VLPs (10 4 /ml) (41,87). In fact, the supernatant of the plasmid transfected 293T cells showed 4 HAU/mL, a titer corresponding to 10 5 PFU/mL of the egg grown influenza C virus (data not shown). However, the supernatant contained only 10 1 to 10 3 EID 50 /ml of infectious viruses (42). The discrepancy between the HA and infectious virus titers could be explained by the presence of large and limited numbers of noninfectious and infectious virus particles, respectively, in the supernatant of the plasmid transfected 293T cells. Neumann et al. reported that in the plasmid driven reverse genetics system, one in to T cells produce the recombinant infectious influenza A virus (5). This means that approximately one in 1,000 cells expresses one set (eight segments) of vrna, leading to the generation of infectious influenza A viruses, and that the remaining 999 in 1,000 cells express less than seven vrna segments. This suggestion seems to apply in the case of influenza C virus reverse genetics: one in 1,000 cells expresses one set (seven segments) of vrna, and the remaining 999 cells express fewer than six RNA segments. Therefore, the above mentioned discrepancy between the HA and infectious virus titers in the supernatant suggests that the remaining 999 cells efficiently produce noninfectious particles. The hypothesis that the 293T cells expressing less than six RNA segments efficiently produce influenza C particles is supported by the previous observations that (i) interaction of HEF with M1 may be sufficient to lead to budding at the cell surface (39, our unpublished result) and (ii) nucleocapsids may not be required to initiate the budding process of the virus (40,88). In other words, in the case of influenza C virion formation, coexpression of M1 and HEF in a transfected cell, even if the ratio of M1 to HEF in the cell is different from that in virus infected cells, may readily lead to particle formation regardless of the presence or absence of nucleocapsids. Furthermore, it is possible that inefficient interaction of M1 with nucleocapsid (39) facilitates the production of noninfectious particles. Alternatively, as a driving force for virion formation, the influenza C virus M1 protein may function more efficiently than the influenza A virus M1 protein. There is a significant difference between the VLP generation efficiencies of influenza C (10 6 /ml) and A (10 4 /ml) viruses (41,87). A comparison of the number of VLPs with that of infectious virus particles between influenza A and C viruses suggests that in the case of influenza C virus generation, cells expressing only one Fig. 5. Hypothesized virion generation mechanism for influenza C and A viruses. 293T cells transfected with plasmid DNAs for reverse genetics are divided into seven (influenza C virus) or eight (influenza A virus) groups according to the number of vrnas expressed in each cell. The length of the white (influenza C virus) and black (influenza A virus) arrows indicates the generation efficiencies of the viruses from the respective cells. In the case of influenza C virus, 293T cells expressing one RNA segment are the most efficient in the production of virions. In contrast, for influenza A virus, cells expressing one set (eight segments) of vrna are the most efficient in the production of infectious virions. 162

7 RNA segment may be the most efficient in the production of virus particles. Based on these findings, we formed a hypothesis explaining the generation of influenza C virus, which is shown in Fig. 5. In the case of influenza A virus, 293T cells expressing one set (eight segments) of vrna produce infectious virions most efficiently. This is consistent with the results reported by Fujii et al., in which cells expressing seven and six segments are less efficient in the production of influenza A viruses (89). In contrast, for influenza C virus, 293T cells expressing one vrna segment may be the most efficient in the production of virions, whereas cells expressing one set (seven segments) of vrna may be the least efficient, resulting in the presence of a large number of noninfectious virions in the supernatant. 8. Prospects for research on influenza C virology A number of influenza C virus proteins are considered to be likely targets for future reverse genetics analyses. CM2 is the second membrane protein of the virus, and its characteristics have been extensively studied (45 48,90). A recombinant influenza C virus lacking CM2 would help elucidate the role of CM2 in the virus replication cycle. NS gene products are also likely candidates for analysis. As mentioned above, the influenza C virus NS2 possesses a nuclear export activity, and the nuclear export signal (NES) is composed of two separate leucine rich domains on the NS2 protein (57). The role of the NES in virus replication, including the role of the individual amino acids in the NES, remains to be elucidated. We have recently reported that NS1 is involved in viral premrna splicing (56), although the functional domain on the NS1 protein has not yet been mapped. Our research group and others have identified the amino acid(s) on the HEF glycoprotein responsible for receptor binding and esterase activity (21,22,27). Furthermore, analysis of transfected cells expressing HEF revealed the role of the individual glycosylation sites of HEF in its proper conformation, biological activities and transport (91). These findings should be further studied in the context of virus replication using recombinant viruses. Phylogenetic analysis showed that the 34 NS genes analyzed were split into two distinct groups, A and B, and suggested that influenza C viruses that acquired group B NS genes through reassortment events dominantly replaced viruses with group A NS genes, forming a stable lineage (54). This finding leads to the hypothesis that the NS gene and/or NS gene product(s) may provide influenza C virus with a determinant for an epidemiological advantage. A comparison of the growth kinetics of recombinants harboring A or B NS genes would clarify the significance of the gene. 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