Jpn. J. Infect. Dis., 63, 157­165, 2010 Review The Molecular Virology and Reverse Genetics of Influenza C Virus Yasushi Muraki1,2* and Seiji Hongo2 1Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa 920­0293; and 2Department of Infectious Diseases, Yamagata University Faculty of Medicine, Yamagata 990­9585, Japan (Received January 18, 2010. Accepted March 29, 2010) CONTENTS 1. Introduction 6. Analysis of the structure­function relationship of 2. Reverse genetics of influenza viruses the M1 protein 3. Molecular virology of influenza C virus 7. Comparison of the generation efficiencies of in­ 4. Epidemiology of influenza C virus fluenza C and A viruses 5. Generation of influenza C virus­like particles and a 8. Prospects for research on influenza C virology recombinant influenza C virus 9. Conclusion SUMMARY: Influenza C virus, an enveloped virus containing seven single­stranded 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 virus­like particle (VLP) generation system and reverse genetics of the virus and succeeded in clarifying the structure­function 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. for the generation of negative­sense RNA viruses, re­ 1. Introduction searchersfacedtheobstacleofprovidingtheviral Reverse genetics, as the term is used in molecular RNA (vRNA) with viral RNA polymerase and nucleo­ virology, describes the generation of viruses possessing protein. In the case of influenza A virus, the viral genome(s) derived from cloned cDNA(s). Of the viruses ribonucleoprotein (vRNP) complex, composed of belonging to the family Orthomyxoviridae,reverse three polymerase subunits (PB2, PB1, and PA), genetics has hitherto been reported for influenza A, in­ nucleoprotein (NP) and vRNA, is minimally required. fluenza B and Thogoto viruses. Recently, our research Initially, by transfecting an artificially reconstituted group, as well as Crescenzo­Chaigne and van der Werf, vRNP complex into eukaryotic cells followed by infec­ reported the successful reverse genetics of influenza C tion with an influenza helper virus, successful recovery virus. of a recombinant influenza virus containing a viral gene In this review, we will first provide an overview of segment derived from the cloned cDNA was reported research on reverse genetics of influenza A virus, and (1,2). then summarize the molecular virology and epidemiolo­ In 1996, Pleschka et al. reported the successful gener­ gy of influenza C virus, including a discussion of ation of a transfectant influenza virus (3). To synthesize presently unresolved issues of the virus. In the latter influenza vRNA in the nucleus, they made use of a part of this review, we will deal with a virus­like particle nucleolar enzyme, RNA polymerase I, in a system that (VLP) generation and reverse genetics of influenza C vi­ had been established by Zobel et al. (4). Cotransfection rus by our research group and provide a hypothesis to of the Pol Iplasmid encoding neuraminidase (NA) explain influenza C virion generation as observed in the vRNA together with viral protein­expressing plasmids established reverse­genetics system. 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 2. Reverse genetics of influenzaviruses in using this method, however, the helper virus­depen­ The genomes of negative­sense RNA viruses, includ­ dent system remained an obstacle to the efficient recov­ ing influenza viruses, are noninfectious. Therefore, ery of the recombinant virus. In 1999, a recombinant influenza A virus was report­ ed to be generated entirely from cloned cDNA for the *Corresponding author: Mailing address: Department of first time (5). The cDNAs of the eight vRNA segments Microbiology, Kanazawa Medical University School of of A/WSN/33 or A/PR/8/34 virus were each cloned Medicine, Uchinada, Ishikawa 920­0293, Japan. Tel: {81­ into the pHH21 vector in negative­sense orientation be­ 76­218­8097, Fax: {81­76­286­3961, E­mail: ymuraki tween the RNA polymerase Ipromoter and terminator kanazawa­med.ac.jp sequences. The resulting eight plasmids were transfected 157 into 293T cells together with four (PB2, PB1, PA, and virus. Yamashita et al. cloned the three largest RNA NP) or nine (PB2, PB1, PA, HA, NP, NA, M1, M2, segments of C/JJ/50 (17). A comparison of the nucleo­ and NS2) viral protein­expressing plasmid DNAs. tide sequences of the segments with those of the influen­ Despite the need for the simultaneous transfection of as za A and B viruses revealed that RNA segments 1 and 2 many as 12 or 17 plasmids into the cells, approximately encode the proteins equivalent to PB2 and PB1 of the 106 to 107 infectious influenza A viruses/mL were pro­ influenza A and B viruses, respectively. The protein en­ duced at 48 to 72 h posttransfection. Thus, a recom­ coded by RNA segment 3 is referred to as P3 rather than binant influenza A virus in which all RNA segments PA, since it does not display any acid charge features at were derived from cDNA clones was generated. a neutral pH. Evidence has been obtained to suggest Improved approaches for the generation of the re­ that the transcription and replication of the influenza C combinant influenza A virus were later reported by virus genome follows the same strategy as that of in­ several research groups (68). Furthermore, influenza B fluenza A virus in that the three proteins form an RNA (911) and Thogoto viruses (12) were successfully gener­ ated in a similar way. Despite all these advances, however, no reverse genetics of influenza C virus was es­ tablished. 3. Molecular virology of influenza C virus Influenza C virus, which belongs to the genus In­ fluenza 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 sin­ gle­stranded RNA segments of negative polarity and en­ codes the following proteins: PB2, PB1, P3, hemag­ glutinin­esterase­fusion (HEF), NP, matrix (M1) pro­ tein, and CM2, as well as the non­structural proteins Fig. 1. Structure of influenza C virus. Influenza C virus has NS1 and NS2 (Figs. 1 and 2). The virus usually causes seven single­stranded RNA segments of negative polarity. The mild upper respiratory illness (14), but it can also cause viral ribonucleoprotein (vRNP) complex is composed of viral RNA (vRNA) and the PB2, PB1, P3, and NP proteins. HEF lower respiratory infections (15,16). forms a spike on the virion. M1 is present beneath the enve­ A number of research groups have studied the struc­ lope. CM2 is the second membrane protein of the virus. A ture and function of the genes and gene products of the 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 positive­sense orientation. Numbers indicate the nucleotide positions along the genes. The lines at the 5? and 3? termini represent the noncoding regions. The V­shaped 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 en­ coded 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 C­terminal region of NS2 (gray area) contains {1 ORF. The C/Ann Arbor/1/50 strain contains a total of 12,906 nucleotides. 158 polymerase complex (1820), although the precise the 374­amino­acid protein, P42, which is cleaved by role(s) of the respective proteins remains to be elucidat­ signal peptidase at an internal cleavage site to give M1' ed. and CM2 (43,44). The biochemical features of CM2, the RNA segment 4 encodes the HEF glycoprotein, which second membrane protein of the virus, are closely simi­ forms a spike on the virus envelope (Fig. 1). The HEF lar to those of M2 (45,46), a proton channel of influenza protein has three biological activities: receptor binding, A virus. Although CM2 expressed in Xenopus laevis oo­ receptor destroying (acetylesterase), and membrane cytes forms an ion channel permeable to Cl| (47) and fusing activities. The receptor of the virus is the 9­O­ CM2 has the ability to modulate the pH of the exocytic acetyl­N­acetylneuraminic acid (Neu5,9Ac2), and the pathway (48), the role of CM2 in the virus replication acetylesterase activity of HEF inactivates the virus cycle remains unclear. M1', composed of the N­terminal receptor by releasing the O­acetyl residues from the C­9 259aminoacidsofP42,isdegradedshortlyafter position of Neu5,9Ac2 (2123). The fusion activity of cleavage through the signal located in the C­terminal 17­ HEF is dependent on the proteolytic cleavage of a amino acid region of the protein (49).