There was a tendency for higher visfatin levels in CHC patients with lower BMI (Huang et al., 2011; Kukla et al., 2010). MS was also negatively correlated with visfatin level in CHC patients. In addition, the extent of glucose abnormalities was not significantly correlated with visfatin level. These observations may imply that visfatin was not related to IR (Pagano et al., 2006). Kukla et al postulated that visfatin may play a dual role as a pro-inflammatory and/or protective factor (Kukla et al., 2010). Taken together, the speculations regarding the role of visfatin in CHC patients include 1) visfatin may not be one of the initiators in the context of metabolic derangements; 2) visfatin may not be involved into the link between obesity and IR in a clinical setting, at least among CHC patients. Therefore, its precise endocrine role deserved to be further clarified in HCV infection.

Several cytokines, particularly adipokines, have been investigated in terms of the link between HCV infection, steatosis, fibrosis, and metabolic abnormalities. These include leptin, adiponectin, apelin, resistin, TNF-α, interleukins, etc(Liu et al., 2005; Marra et al., 2009). It is informative and helpful for the elucidation of pathogenic mechanisms to assess the interaction between related biomarkers and clinical profiles of HCV infection, such as metabolic, virological, and histological factors.

#### **5. Conclusion**

Metabolic disorders are characteristic in patients with HCV infection. Besides established liver injury, the multiple extrahepatic metabolic disorders lead to a certain extent of impact on the disease activity, disease course, clinical outcomes, and treatment efficacy of current therapy. Enormous challenges for patient management have been arised in parallel with the occurrence of these metabolic disorders. Although the precise mechanisms contributing to each aspect of metabolic abnormalities are not fully clarified, inspiring data and studies during the past decades have much enhanced our knowledge of this unique link between a viral hepatitis disease and many common metabolic manifestations. With the rapid progression of new therapy for CHC, i.e. direct acting antivirals, in the past few years, the interaction between HCV infection and metabolic disorders will become clearer. Meanwhile, recent promisng data based on genome-wide association studies largely explore our understanding of the impact of host susceptibility on the treatment efficacy of CHC. It may also pave the way for the future study regarding the genetic and/or proteomic aspects of metabolic abnormalities in HCV infection. On the other side, management of the metabolic disorders mainly depends on both pharmaceutical intervention and lifestyle modifications, such as exercise, diet control and weight reduction for T2DM and dyslipemia. Whether these interventions play a role in the disease course and prognosis of CHC patients deserves to be elucidated in the future.

#### **6. Acknowledgments**

The authors thank secretary help from Taiwan Liver Research Foundation (TLRF).

#### **7. References**

50 Molecular Virology

expressed by visceral adipose tissue compared with subcutaneous fat(Fukuhara et al., 2005).  However, during the past years, contradictory results regarding waist circumference, glucose level, IR, visceral and subcutaneous adipose tissues have also been emerged in a clinical setting(Sommer et al., 2008). Berndt et al demonstrated that serum visfatin level did not correlate with visceral fat mass and that mRNA expression of the gene encoding visfatin was similar in visceral and subcutaneous adipose tissue (Berndt et al., 2005). Pagano et al demonstrated that serum visfatin level was decreased by approximately 50% in obese patients compared with controls. Furthermore, a negative correlation was found between serum visfatin level and BMI (Pagano et al., 2006). In the aspect of HCV infection, CHC patients tended to have a higher visfatin level than that of healthy controls. Serum visfatin level was correlated significantly with histological activity index scores and fibrosis stages, namely, disease severity in CHC patients (Huang et al., 2011). It hence implied that visfatin could be a potential biomarker for prediction of disease severity in HCV infection. The concordant results in different entities of liver diseases provided evidence for previous experimental studies indicating that visfatin was involved into inflammatory process network (Lim et al., 2008). On the other side, the studies addressing the correlation between visfatin and HCV genotypes were not concordant (Kukla et al., 2010). There was no significant correlation between visfatin and viral load, and treatment response to PegIFN/ribavirin therapy. Further study is warranted to clarify in which a concealed player or a bystander of the necroinflammatory and fibrogenetic

There was a tendency for higher visfatin levels in CHC patients with lower BMI (Huang et al., 2011; Kukla et al., 2010). MS was also negatively correlated with visfatin level in CHC patients. In addition, the extent of glucose abnormalities was not significantly correlated with visfatin level. These observations may imply that visfatin was not related to IR (Pagano et al., 2006). Kukla et al postulated that visfatin may play a dual role as a pro-inflammatory and/or protective factor (Kukla et al., 2010). Taken together, the speculations regarding the role of visfatin in CHC patients include 1) visfatin may not be one of the initiators in the context of metabolic derangements; 2) visfatin may not be involved into the link between obesity and IR in a clinical setting, at least among CHC patients. Therefore, its precise

Several cytokines, particularly adipokines, have been investigated in terms of the link between HCV infection, steatosis, fibrosis, and metabolic abnormalities. These include leptin, adiponectin, apelin, resistin, TNF-α, interleukins, etc(Liu et al., 2005; Marra et al., 2009). It is informative and helpful for the elucidation of pathogenic mechanisms to assess the interaction between related biomarkers and clinical profiles of HCV infection, such as

Metabolic disorders are characteristic in patients with HCV infection. Besides established liver injury, the multiple extrahepatic metabolic disorders lead to a certain extent of impact on the disease activity, disease course, clinical outcomes, and treatment efficacy of current therapy. Enormous challenges for patient management have been arised in parallel with the

endocrine role deserved to be further clarified in HCV infection.

metabolic, virological, and histological factors.

**5. Conclusion**

scenarios visfatin behaves.

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**4**

*Japan*

Yasushi Muraki

*Department of Microbiology*

**Influenza C Virus: Structure and**

*Kanazawa Medical University School of Medicine*

**Function of M Gene and Its Products**

Influenza C virus (Fig.1), which belongs to the genus *Influenza C Virus* of the family *Orthomyxoviridae*, was first isolated from a patient with respiratory illness in 1947 (Taylor, 1949). It is widely distributed throughout the world and the majority of humans acquire antibodies to the virus early in life (Homma et al., 1982; Nishimura et al., 1987). The virus usually causes a mild upper respiratory illness (Katagiri et al., 1983), but can also cause lower respiratory infections such as bronchitis and pneumonia (Moriuchi et al., 1991; Matsuzaki et al., 2006). Recently, a case of acute encephalopathy associated with influenza C virus infection has been reported for the first time (Takayanagi et al., 2009). Although influenza C virus is isolated infrequently due to lack of facilities equipped with the resources for performing efficient virus isolation, recurrent infection with this virus occurs frequently in children as well as in adults (Homma et al., 1982; Katagiri et al., 1983, 1987;

The genome of influenza C virus consists of seven RNA segments of negative polarity, each of which encodes three polymerase proteins (PB2, PB1, and P3), hemagglutinin-esterasefusion (HEF) glycoprotein, nucleoprotein (NP), matrix (M1) protein and CM2, and two nonstructural proteins (NS1 and NS2/NEP) (Palese & Shaw, 2007). PB2, PB1 and P3 are subunits of the RNA polymerase of the virus (Crescenzo-Chaigne et al., 1999; Crescenzo-Chaigne & van der Werf, 2001; Nagele & Meier-Ewert 1984; Yamashita et al., 1989). HEF, which has receptor-binding, receptor-destroying and fusion activities, forms a spike on the virion (Herrler & Klenk, 1991). NP participates in forming ribonucleoproteins (RNPs) with viral RNA (vRNA), PB2, PB1 and P3 (Crescenzo-Chaigne et al., 1999; Crescenzo-Chaigne & van der Werf, 2001; Nakada et al., 1984; Sugawara et al., 1991; 2006). M1 is abundantly present beneath the envelope, which gives rigidity to the virion. CM2 is the second membrane protein of the virus. NS1 is involved in viral mRNA splicing (Muraki et al. 2010), and NS2/NEP is a nuclear export protein (Paragas et al., 2001) and is incorporated into the

In this chapter, the author will focus on the M gene and M gene products and describe how they contribute to the replication of influenza C virus. First, the author will show the M gene structure, including its coding strategy. Second, the author will mention the characteristics

**1. Introduction**

Matsuzaki et al., 1990).

virions (Kohno et al. 2009).

implications in the hepatitis C virus hyperendemic areas and a tertiary referral center in Taiwan. *J Med Virol* 65(1), 58-65.

## **Influenza C Virus: Structure and Function of M Gene and Its Products**

Yasushi Muraki

*Department of Microbiology Kanazawa Medical University School of Medicine Japan*

#### **1. Introduction**

62 Molecular Virology

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Influenza C virus (Fig.1), which belongs to the genus *Influenza C Virus* of the family *Orthomyxoviridae*, was first isolated from a patient with respiratory illness in 1947 (Taylor, 1949). It is widely distributed throughout the world and the majority of humans acquire antibodies to the virus early in life (Homma et al., 1982; Nishimura et al., 1987). The virus usually causes a mild upper respiratory illness (Katagiri et al., 1983), but can also cause lower respiratory infections such as bronchitis and pneumonia (Moriuchi et al., 1991; Matsuzaki et al., 2006). Recently, a case of acute encephalopathy associated with influenza C virus infection has been reported for the first time (Takayanagi et al., 2009). Although influenza C virus is isolated infrequently due to lack of facilities equipped with the resources for performing efficient virus isolation, recurrent infection with this virus occurs frequently in children as well as in adults (Homma et al., 1982; Katagiri et al., 1983, 1987; Matsuzaki et al., 1990).

The genome of influenza C virus consists of seven RNA segments of negative polarity, each of which encodes three polymerase proteins (PB2, PB1, and P3), hemagglutinin-esterasefusion (HEF) glycoprotein, nucleoprotein (NP), matrix (M1) protein and CM2, and two nonstructural proteins (NS1 and NS2/NEP) (Palese & Shaw, 2007). PB2, PB1 and P3 are subunits of the RNA polymerase of the virus (Crescenzo-Chaigne et al., 1999; Crescenzo-Chaigne & van der Werf, 2001; Nagele & Meier-Ewert 1984; Yamashita et al., 1989). HEF, which has receptor-binding, receptor-destroying and fusion activities, forms a spike on the virion (Herrler & Klenk, 1991). NP participates in forming ribonucleoproteins (RNPs) with viral RNA (vRNA), PB2, PB1 and P3 (Crescenzo-Chaigne et al., 1999; Crescenzo-Chaigne & van der Werf, 2001; Nakada et al., 1984; Sugawara et al., 1991; 2006). M1 is abundantly present beneath the envelope, which gives rigidity to the virion. CM2 is the second membrane protein of the virus. NS1 is involved in viral mRNA splicing (Muraki et al. 2010), and NS2/NEP is a nuclear export protein (Paragas et al., 2001) and is incorporated into the virions (Kohno et al. 2009).

In this chapter, the author will focus on the M gene and M gene products and describe how they contribute to the replication of influenza C virus. First, the author will show the M gene structure, including its coding strategy. Second, the author will mention the characteristics

Influenza C Virus: Structure and Function of M Gene and Its Products 65

The mRNA for M1 represents the major M gene-specific mRNA species in virus-infected cells. It is of interest that the splicing pattern of the influenza C virus M gene is different from that of influenza A virus M gene, in that the influenza A virus M1 protein, a major product of the

An open reading frame (ORF) in the influenza C virus M gene could potentially code for a protein of 374 amino acids, and small quantities of an mRNA collinear with RNA segment 6 was actually detected since a faint signal corresponding to the mRNA species was observed by S1 analysis (Yamashita et al., 1988). At that moment, however, the significance of the

Analysis of the nucleotide sequences of the M gene from five influenza C virus isolates, including C/Ann Arbor/1/50, revealed that the gene contains a single ORF capable of coding for a 374-amino-acid protein (Hongo et al., 1994; Yamashita et al., 1988). Therefore, attempts was made to identify the unspliced mRNA of the M gene, and the mRNA was found to be synthesized in 1/10 amount of the spliced mRNA in the virus-infected cells (Hongo et al., 1994). To identify the protein encoded by the unspliced mRNA, whose approximate molecular weight is 42,000, the antiserum against the glutathione S-transferase fusion protein containing the extra C-terminal domain of the protein (Fig. 2) was prepared. Unexpectedly, immunoprecipitation experiments with the antiserum identified a protein of Mr ~18,000 in virus-infected cells (Hongo et al., 1994). The authors designated the protein as CM2, a second protein encoded by the influenza C virus M gene, and identified three forms of CM2 (CM2o, CM2a, and CM2b) depending on the moieties of glycosylation (Hongo et al.,

A previously unrecognized 374-amino-acid protein was identified in virus-infected cells using the antiserum against CM2, and the protein was named as P42 according to the molecular weight. P42 is demonstrated to be modified by the addition of a high-mannose oligosaccharide chain to generate P44. The tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. Thus, the authors concluded that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA

At that moment, the translational mechanism of CM2 was still unclear. It was reported previously that the P42 is an integral membrane protein having two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favourable for cleavage by signal peptidase. To examine the possibility that P42 is cleaved by signal peptidase to yield CM2, a series of mutant M gene cDNAs were constructed and transfected into cultured cells (Hongo et al., 1999; Pekosz & Lamb, 1998). As a result, biosynthesis of CM2 has been demonstrated to proceed in the following manner; (i) Unspliced mRNA from RNA segment 6 is first translated into a 374-amino-acid protein, P42. (ii) Cotranslationally or immediately after the completion of translation, P42 begins to be inserted into the endoplasmic reticulum with the aid of the first hydrophobic domain, composed of amino acid residues 241 to 252. (iii) Translocation of P42 through the membrane is halted by the presence of the second hydrophobic domain, consisting of residues 287 to 318. (iv) P42 is then N-glycosylated at Asn residue 270, generating P44. (v)

segment 6 mRNA and its N-glycosylated form, respectively (Hongo et al., 1998).

gene, is coded for by an unspliced mRNA, not spliced mRNA (Palese & Shaw, 2007).

collinear mRNA species remained unknown.

**2.2 CM2 protein**

1994).

of M1 and CM2 proteins. Finally, the author will describe recent findings on M1 and CM2 proteins with regard to their roles in virus replication.

Fig. 1. Structure of influenza C virus.

The influenza C virus contains seven single-stranded RNA segments of negative polarity. The viral ribonucleoprotein (vRNP) complex is composed of viral RNA (black line), PB2 (open circle), PB1 (open rectangle), PA (open triangle) and NP (closed circle) proteins. The structure of vRNP is depicted only for the RNA Segment 7 for simplicity. HEF is a spike protein of the virus, forming homotrimers on the envelope. M1 is located beneath the envelope. CM2 is the second membrane protein that is present as a homotetramer. A small amount of NS2/NEP is incorporated into the virions.

#### **2. Coding strategy of influenza C virus M gene**

The influenza C virus RNA segment 6 (M gene) is 1,180 nucleotides in length and encodes a 242-amino-acid matrix protein (M1) and 115-amino-acid CM2 protein. In this section, the author will describe the coding strategy of the M gene, including the brief history of M1 and CM2 discovery.

#### **2.1 M1 protein**

Yamashita et al. determined the nucleotide sequence of C/Ann Arbor/1/50 M gene and provided evidence that a 242-amino-acid M1 protein is encoded by a spliced mRNA of the gene (Yamashita et al., 1988). The spliced mRNA of the gene lacks a region from nucleotides 754 to 981 due to the introduction of the stop codon (TGA) as a result of splicing (Fig. 2). The splicing event itself introduced the stop codon TGA, with the TG coming from the 5' splicing site and the A coming from the 3' splicing site.

The mRNA for M1 represents the major M gene-specific mRNA species in virus-infected cells. It is of interest that the splicing pattern of the influenza C virus M gene is different from that of influenza A virus M gene, in that the influenza A virus M1 protein, a major product of the gene, is coded for by an unspliced mRNA, not spliced mRNA (Palese & Shaw, 2007).

An open reading frame (ORF) in the influenza C virus M gene could potentially code for a protein of 374 amino acids, and small quantities of an mRNA collinear with RNA segment 6 was actually detected since a faint signal corresponding to the mRNA species was observed by S1 analysis (Yamashita et al., 1988). At that moment, however, the significance of the collinear mRNA species remained unknown.

#### **2.2 CM2 protein**

64 Molecular Virology

of M1 and CM2 proteins. Finally, the author will describe recent findings on M1 and CM2

The influenza C virus contains seven single-stranded RNA segments of negative polarity. The viral ribonucleoprotein (vRNP) complex is composed of viral RNA (black line), PB2 (open circle), PB1 (open rectangle), PA (open triangle) and NP (closed circle) proteins. The structure of vRNP is depicted only for the RNA Segment 7 for simplicity. HEF is a spike protein of the virus, forming homotrimers on the envelope. M1 is located beneath the envelope. CM2 is the second membrane protein that is present as a homotetramer. A small

The influenza C virus RNA segment 6 (M gene) is 1,180 nucleotides in length and encodes a 242-amino-acid matrix protein (M1) and 115-amino-acid CM2 protein. In this section, the author will describe the coding strategy of the M gene, including the brief history of M1 and

Yamashita et al. determined the nucleotide sequence of C/Ann Arbor/1/50 M gene and provided evidence that a 242-amino-acid M1 protein is encoded by a spliced mRNA of the gene (Yamashita et al., 1988). The spliced mRNA of the gene lacks a region from nucleotides 754 to 981 due to the introduction of the stop codon (TGA) as a result of splicing (Fig. 2). The splicing event itself introduced the stop codon TGA, with the TG coming from the 5'

HEF

CM2

M1

vRNP

NS2

Lipid bilayer

vRNA PB2 PB1 P3 NP

proteins with regard to their roles in virus replication.

Fig. 1. Structure of influenza C virus.

CM2 discovery.

**2.1 M1 protein**

amount of NS2/NEP is incorporated into the virions.

**2. Coding strategy of influenza C virus M gene**

splicing site and the A coming from the 3' splicing site.

Analysis of the nucleotide sequences of the M gene from five influenza C virus isolates, including C/Ann Arbor/1/50, revealed that the gene contains a single ORF capable of coding for a 374-amino-acid protein (Hongo et al., 1994; Yamashita et al., 1988). Therefore, attempts was made to identify the unspliced mRNA of the M gene, and the mRNA was found to be synthesized in 1/10 amount of the spliced mRNA in the virus-infected cells (Hongo et al., 1994). To identify the protein encoded by the unspliced mRNA, whose approximate molecular weight is 42,000, the antiserum against the glutathione S-transferase fusion protein containing the extra C-terminal domain of the protein (Fig. 2) was prepared. Unexpectedly, immunoprecipitation experiments with the antiserum identified a protein of Mr ~18,000 in virus-infected cells (Hongo et al., 1994). The authors designated the protein as CM2, a second protein encoded by the influenza C virus M gene, and identified three forms of CM2 (CM2o, CM2a, and CM2b) depending on the moieties of glycosylation (Hongo et al., 1994).

A previously unrecognized 374-amino-acid protein was identified in virus-infected cells using the antiserum against CM2, and the protein was named as P42 according to the molecular weight. P42 is demonstrated to be modified by the addition of a high-mannose oligosaccharide chain to generate P44. The tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. Thus, the authors concluded that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA segment 6 mRNA and its N-glycosylated form, respectively (Hongo et al., 1998).

At that moment, the translational mechanism of CM2 was still unclear. It was reported previously that the P42 is an integral membrane protein having two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favourable for cleavage by signal peptidase. To examine the possibility that P42 is cleaved by signal peptidase to yield CM2, a series of mutant M gene cDNAs were constructed and transfected into cultured cells (Hongo et al., 1999; Pekosz & Lamb, 1998). As a result, biosynthesis of CM2 has been demonstrated to proceed in the following manner; (i) Unspliced mRNA from RNA segment 6 is first translated into a 374-amino-acid protein, P42. (ii) Cotranslationally or immediately after the completion of translation, P42 begins to be inserted into the endoplasmic reticulum with the aid of the first hydrophobic domain, composed of amino acid residues 241 to 252. (iii) Translocation of P42 through the membrane is halted by the presence of the second hydrophobic domain, consisting of residues 287 to 318. (iv) P42 is then N-glycosylated at Asn residue 270, generating P44. (v)

Influenza C Virus: Structure and Function of M Gene and Its Products 67

The biochemical characteristics of CM2 in virus-infected cells were precisely determined, and as a result, CM2 is demonstrated to be the counterpart of the influenza A virus M2 protein (Hongo et al., 1997; Pekosz & Lamb, 1997; Tada et al., 1998). CM2 is a type III integral membrane protein that is oriented in membranes with a 23-amino-acid N-terminal extracellular domain, a 23-amino-acid transmembrane domain and a 69-amino-acid Cterminal cytoplasmic domain. It forms homodimer and homotetramer, and is phosphorylated, palmitoylated and N-glycosylated, and is incorporated into progeny virions. Site-specific mutational analyses of the M gene identified the amino acid sites for the posttranslational modifications on CM2 molecules as follows; i) an asparagine residue 11 is an N-glycosylation site, ii) cysteines at residue 1, 6 and 20 are all involved in disulfide bond formation, iii) a cysteine residue at 65 is palmitoylated through labile thioester linkage, and iv) serine residues at 78, 103, 108 and a proline at 104 are phosphorylation sites (Li et al.,

CM2 has also been investigated from an electrophysiological point of view. A number of studies have shown that the CM2 protein appears to have ion channel activities. CM2 forms a voltage-activated ion channel permeable to Cl<sup>−</sup> (Hongo et al., 2004). When co-expressed with a pH-sensitive hemagglutinin (HA) from influenza A virus, the CM2 protein has a capacity to reduce the acidity of the exocytic pathway and reduce conversion of the pHsensitive HA to its low pH conformation during transport to the cell surface, suggesting that CM2 is permeable to H+ (Betáková & Hay, 2007). Preliminary electrophysiological studies of CM2-expressing mouse erythroleukemia cells have identified Na+-activated proton permeability in addition to the low pH-activated Cl<sup>−</sup> permeability (Muraki & Hay, 2009; Muraki et al., personal communications). However, the relationship between the channel function and the role(s) of CM2 in the virus replication (see below) remains to be clarified.

The M1 protein of influenza C virus is involved in virion morphogenesis. Nishimura et al. reported that cord-like structures (CLSs) which had lengths up to ~500 μm or greater were protruding from the surface of C/Yamagata/1/88-infected HMV-II cells (Nishimura et al., 1990). Electron microscopic analysis showed that CLS consists of numerous filamentous particles in the process of budding, each of which is covered with a layer of surface projections and aggregated with their long axes. Further analyses using 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. Comparison of the M gene sequences of influenza C virus strains, including C/Yamagata/1/88 and C/Taylor/1233/47, suggested that either or both of amino acid changes at positions 24 and 133 of the M1 protein were responsible for differences in cord-forming ability between the

The amino acid on the M1 protein that is responsible for CLS formation and virion morphology was determined. Upon establishment of influenza C virus-like particle (VLP) generation system, Muraki et al. observed that CLS was protruding from the 293T cells

**4. Role of the M gene-products in virus replication**

**3.2 CM2 protein**

2001; Pekosz & Lamb, 1997).

**4.1 M1 protein**

two strains (Nishimura et al., 1994).

Either before or after the addition of an oligosaccharide chain, P42/P44 is cleaved by signal peptidase at the C-terminal side of Ala residue 259, producing the M1' and CM2 proteins, composed of the N-terminal 259 amino acids and the C-terminal 115 amino acids, respectively.

Fig. 2. Coding strategy of influenza C virus M gene.

RNA segments 6 (M gene) of C/Ann Arbor/1/50 is shown in positive-sense orientation. Numbers indicate the nucleotide positions along the genes. The lines at the 5' and 3' termini represent the non-coding regions. The boxes represent the coding region of viral proteins encoded by the gene. The intron is shown by V-shaped lines. M1 is encoded by a spliced mRNA (upper panel), into which the stop codon TGA is introduced at 752, 753 and 982, as a result of splicing. The P42 protein (M1'+CM2), encoded by a collinear transcript of the gene (lower panel), is cleaved by signal peptidase at an internal cleavage site (closed triangle) to generate M1' and CM2.

#### **3. Characteristics of the M1 and CM2 proteins**

#### **3.1 M1 protein**

The influenza C virion contains three major structural polypeptides: a large glycoprotein gp88 (HEF), a nucleoprotein (NP) and matrix protein (M1) (Palese & Shaw, 2007). Furthermore, Yokota et al. identified the M1 protein synthesized in C/Ann Arbor/1/50 infected MDCK cells (Yokota et al., 1981). To characterize the protein, the monoclonal antibodies against M1 have been produced (Sugawara et al., 1991). Using a panel of the nine MAbs against M1, the M1 protein was shown to contain two non-overlapping antigenic regions that are highly resistant to conformational changes, and to exhibit no antigenic variations among 23 influenza C virus strains isolated over 41 year period. The C/Ann Arbor/1/50-infected cells were examined for the localization of the M1 protein using one of these MAbs (L2), and as a result, M1 is shown to be localized in the nucleus at 24 h postinfection. The latter finding provided evidence for the requirement of nuclear function for influenza C virus replication. In addition, the association of M1 with the nucleoli was observed in the C/Yamagata/1/88-infected cells, a phenomenon that remains to be elucidated with respect to viral replication (Sugawara et al., 1991).

#### **3.2 CM2 protein**

66 Molecular Virology

Either before or after the addition of an oligosaccharide chain, P42/P44 is cleaved by signal peptidase at the C-terminal side of Ala residue 259, producing the M1' and CM2 proteins, composed of the N-terminal 259 amino acids and the C-terminal 115 amino acids,

751

RNA segments 6 (M gene) of C/Ann Arbor/1/50 is shown in positive-sense orientation. Numbers indicate the nucleotide positions along the genes. The lines at the 5' and 3' termini represent the non-coding regions. The boxes represent the coding region of viral proteins encoded by the gene. The intron is shown by V-shaped lines. M1 is encoded by a spliced mRNA (upper panel), into which the stop codon TGA is introduced at 752, 753 and 982, as a result of splicing. The P42 protein (M1'+CM2), encoded by a collinear transcript of the gene (lower panel), is cleaved by signal peptidase at an internal cleavage site (closed triangle) to

The influenza C virion contains three major structural polypeptides: a large glycoprotein gp88 (HEF), a nucleoprotein (NP) and matrix protein (M1) (Palese & Shaw, 2007). Furthermore, Yokota et al. identified the M1 protein synthesized in C/Ann Arbor/1/50 infected MDCK cells (Yokota et al., 1981). To characterize the protein, the monoclonal antibodies against M1 have been produced (Sugawara et al., 1991). Using a panel of the nine MAbs against M1, the M1 protein was shown to contain two non-overlapping antigenic regions that are highly resistant to conformational changes, and to exhibit no antigenic variations among 23 influenza C virus strains isolated over 41 year period. The C/Ann Arbor/1/50-infected cells were examined for the localization of the M1 protein using one of these MAbs (L2), and as a result, M1 is shown to be localized in the nucleus at 24 h postinfection. The latter finding provided evidence for the requirement of nuclear function for influenza C virus replication. In addition, the association of M1 with the nucleoli was observed in the C/Yamagata/1/88-infected cells, a phenomenon that remains to be

1150

CM2(115)

TG A

982

802/803

1180

respectively.

1

26

generate M1' and CM2.

**3.1 M1 protein**

M1'(259)

Fig. 2. Coding strategy of influenza C virus M gene.

**3. Characteristics of the M1 and CM2 proteins**

elucidated with respect to viral replication (Sugawara et al., 1991).

M1 (242)

26

The biochemical characteristics of CM2 in virus-infected cells were precisely determined, and as a result, CM2 is demonstrated to be the counterpart of the influenza A virus M2 protein (Hongo et al., 1997; Pekosz & Lamb, 1997; Tada et al., 1998). CM2 is a type III integral membrane protein that is oriented in membranes with a 23-amino-acid N-terminal extracellular domain, a 23-amino-acid transmembrane domain and a 69-amino-acid Cterminal cytoplasmic domain. It forms homodimer and homotetramer, and is phosphorylated, palmitoylated and N-glycosylated, and is incorporated into progeny virions. Site-specific mutational analyses of the M gene identified the amino acid sites for the posttranslational modifications on CM2 molecules as follows; i) an asparagine residue 11 is an N-glycosylation site, ii) cysteines at residue 1, 6 and 20 are all involved in disulfide bond formation, iii) a cysteine residue at 65 is palmitoylated through labile thioester linkage, and iv) serine residues at 78, 103, 108 and a proline at 104 are phosphorylation sites (Li et al., 2001; Pekosz & Lamb, 1997).

CM2 has also been investigated from an electrophysiological point of view. A number of studies have shown that the CM2 protein appears to have ion channel activities. CM2 forms a voltage-activated ion channel permeable to Cl<sup>−</sup> (Hongo et al., 2004). When co-expressed with a pH-sensitive hemagglutinin (HA) from influenza A virus, the CM2 protein has a capacity to reduce the acidity of the exocytic pathway and reduce conversion of the pHsensitive HA to its low pH conformation during transport to the cell surface, suggesting that CM2 is permeable to H+ (Betáková & Hay, 2007). Preliminary electrophysiological studies of CM2-expressing mouse erythroleukemia cells have identified Na+-activated proton permeability in addition to the low pH-activated Cl<sup>−</sup> permeability (Muraki & Hay, 2009; Muraki et al., personal communications). However, the relationship between the channel function and the role(s) of CM2 in the virus replication (see below) remains to be clarified.

#### **4. Role of the M gene-products in virus replication**

#### **4.1 M1 protein**

The M1 protein of influenza C virus is involved in virion morphogenesis. Nishimura et al. reported that cord-like structures (CLSs) which had lengths up to ~500 μm or greater were protruding from the surface of C/Yamagata/1/88-infected HMV-II cells (Nishimura et al., 1990). Electron microscopic analysis showed that CLS consists of numerous filamentous particles in the process of budding, each of which is covered with a layer of surface projections and aggregated with their long axes. Further analyses using 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. Comparison of the M gene sequences of influenza C virus strains, including C/Yamagata/1/88 and C/Taylor/1233/47, suggested that either or both of amino acid changes at positions 24 and 133 of the M1 protein were responsible for differences in cord-forming ability between the two strains (Nishimura et al., 1994).

The amino acid on the M1 protein that is responsible for CLS formation and virion morphology was determined. Upon establishment of influenza C virus-like particle (VLP) generation system, Muraki et al. observed that CLS was protruding from the 293T cells

Influenza C Virus: Structure and Function of M Gene and Its Products 69

A recombinant influenza C virus lacking CM2 palmitoylation, rCM2-C65A, was successfully rescued (Muraki et al., 2011). The rCM2-C65A, in which the cysteine at residue 65 of CM2 was substituted to alanine, grew as efficiently as did the parental recombinant virus in cultured cells and embryonated chicken eggs. The transport, maturation and localization of HEF, NP, M1 and CM2 in the cells infected with rCM2-C65A were virtually identical to those of the parental virus-infected cells. These findings suggest that CM2 palmitoylation is not required for virus replication. The fact that a recombinant lacking CM2 palmitoylation could be rescued suggests that CM2 mutant(s) at posttranslational site(s) is available for the

The ion channel proteins of influenza A and B viruses play critical roles in the viral replication. The influenza A virus M2 protein functions as a proton channel during the entry of the virus into cells by allowing the acidification of the virion interior (Palese & Shaw, 2007). Recently, the M2 proton-selective ion channel function mediates virus budding (Rossman et al., 2010b; 2011). The M2 cytoplasmic tail also plays a role in progeny virus production and virion morphology (Chen et al., 2008; Iwatsuki-Horimoto et al. 2006; McCown & Pekosz, 2005; Rossman et al., 2010a; Zhang et al., 2000). The influenza B virus BM2 protein has a proton channel activity (Mould et al., 2003), and is involved in the incorporation of the virus genome into virions (Imai et al., 2004; 2008). Thus CM2 needs to

As mentioned above, P42, encoded by a collinear transcript of the RNA segment 6 (M gene), is cleaved by signal peptidase to generate CM2, composed of the C-terminal 115 amino acids, in addition to the M1' protein, composed of the N-terminal 259 amino acids (Hongo S et al., 1999; Pekosz & Lamb 1998). It is of interest to clarify the role(s) of P42 and M1' in virus replication. The characteristics of the P42 and M1' (or p31) proteins were precisely determined as follow. The M1' protein was found to undergo rapid degradation after cleavage from P42, since addition of the 26S proteasome inhibitor lactacystin to the cells infected with influenza C virus or transfected with M gene cDNA drastically reduced M1' degradation (Pekosz & Lamb 2000). In addition, the hydrophobic nature, not the specific amino acid sequence, of the 17-amino-acid C terminus of M1' was demonstrated to act as the signal for targeting the protein to membranes and for degradation (Pekosz & Lamb 2000). Thus, M1' seems to be

A series of mutant M gene cDNAs were transfected into COS-1 cells to examine the characteristics of P42 (Li et al., 2004). P44, the N-glycosylated form of P42, forms disulfidelinked dimers and tetramers. P44 is transported to the Golgi apparatus, but not to the trans-Golgi, since P44 is sensitive to endoglycosidase H. P44 and P42 are unstable irrespective of N-glycosylation or oligomerization. The addition of lactacystin prevented the degradation of P42 as well as M1', but not that of P44 efficiently, suggesting that P44 is degraded by another protease besides the 26S proteasome (Li et al., 2004). The role(s) of P42/P44 in virus

In influenza A virus-infected cells, the regulatory machanism for viral mRNA splicing has been extensively investigated. It has been reported that the steady-state level of spliced viral

analysis of CM2 role(s) in the virus replication.

be further investigated from these points of view.

**4.3 M1' and P42**

dispensable to virus replication.

replication remains to be clarified.

**5. Regulation of M1 and CM2 synthesis**

transfected with a set of plasmid DNAs for VLP generation (Muraki et al., 2004). CLSs were observed on the 293T cells transfected with the plasmid for M1 of C/Ann Arbor/1/50, whereas CLSs were not observed when the plasmid for M1 of C/Ann Arbor/1/50 was replaced with that of C/Taylor/1233/47. Expression of the M1 protein possessing alanine or threonine at residue 24 together with the other virus components resulted in the generation of filamentous or spherical VLPs, respectively. These findings indicate that an amino acid at residue 24 of the M1 protein is a determinant for virion morphology.

The above finding obtained from VLP generation system has been confirmed using a recombinant virus generated by reverse genetics. The parental recombinant influenza C virus, which has the consensus sequences of C/Ann Arbor/1/50 (the residue 24 of M1 is alanine), exhibited a filamentous morphology. In contrast, a recombinant mutant virus, rMG96A, which has an Ala→Thr mutation at residue 24 of M1, exhibited spherical morphology. The mutant M1 protein had lower membrane affinity than did the wild type M1, suggesting that the difference in the affinity affects the virion morphology (Muraki et al., 2007). Furthermore, the flotation analysis showed that budding of influenza C virus does not occur from the lipid raft domain of the plasma membrane. These studies indicate that M1 plays an important role in virus budding, and imply that a region around the residue 24 of M1 may represent a late domain of the protein and that budding mechanism of influenza C virus differs from that of influenza A virus.

#### **4.2 CM2 protein**

The fact that the biochemical characteristics of CM2 are closely similar to those of influenza A virus M2 and that CM2 seems to have permeability to proton suggests that CM2 is also crucial in influenza C virus replication. To clarify the role(s) of CM2 in the influenza C virus replication cycle, attempts were made to generate a recombinant CM2-deficient virus using reverse genetics. However, infectious recombinants lacking CM2 have not been rescued to date (Furukawa et al., 2011), which highly suggests that CM2 is essential to influenza C virus replication.

Using VLP generation system, evidence was obtained that CM2 is involved in the packaging and uncoating processes. The CM2-deficient influenza C VLPs were successfully generated and the amount of GFP-vRNA in the VLPs was quantified. The data showed that the CM2-deficient VLPs contain approximately 37% of the vRNA found in wild-type VLPs, although no significant differences were detected in the expression levels of viral components in VLP-producing cells as well as in the number and morphology of the generated VLPs (Furukawa et al., 2011). This finding suggests that CM2 is involved in the mini-genome packaging into VLPs. In addition, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced GFP expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of GFP-vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner (Furukawa et al., 2011). Thus, CM2 appears to play a role in the packaging and uncoating processes of the virus replication cycle. However, whether and how the CM2 channel activities relate to the roles remains unclear.

A recombinant influenza C virus lacking CM2 palmitoylation, rCM2-C65A, was successfully rescued (Muraki et al., 2011). The rCM2-C65A, in which the cysteine at residue 65 of CM2 was substituted to alanine, grew as efficiently as did the parental recombinant virus in cultured cells and embryonated chicken eggs. The transport, maturation and localization of HEF, NP, M1 and CM2 in the cells infected with rCM2-C65A were virtually identical to those of the parental virus-infected cells. These findings suggest that CM2 palmitoylation is not required for virus replication. The fact that a recombinant lacking CM2 palmitoylation could be rescued suggests that CM2 mutant(s) at posttranslational site(s) is available for the analysis of CM2 role(s) in the virus replication.

The ion channel proteins of influenza A and B viruses play critical roles in the viral replication. The influenza A virus M2 protein functions as a proton channel during the entry of the virus into cells by allowing the acidification of the virion interior (Palese & Shaw, 2007). Recently, the M2 proton-selective ion channel function mediates virus budding (Rossman et al., 2010b; 2011). The M2 cytoplasmic tail also plays a role in progeny virus production and virion morphology (Chen et al., 2008; Iwatsuki-Horimoto et al. 2006; McCown & Pekosz, 2005; Rossman et al., 2010a; Zhang et al., 2000). The influenza B virus BM2 protein has a proton channel activity (Mould et al., 2003), and is involved in the incorporation of the virus genome into virions (Imai et al., 2004; 2008). Thus CM2 needs to be further investigated from these points of view.

#### **4.3 M1' and P42**

68 Molecular Virology

transfected with a set of plasmid DNAs for VLP generation (Muraki et al., 2004). CLSs were observed on the 293T cells transfected with the plasmid for M1 of C/Ann Arbor/1/50, whereas CLSs were not observed when the plasmid for M1 of C/Ann Arbor/1/50 was replaced with that of C/Taylor/1233/47. Expression of the M1 protein possessing alanine or threonine at residue 24 together with the other virus components resulted in the generation of filamentous or spherical VLPs, respectively. These findings indicate that an amino acid at

The above finding obtained from VLP generation system has been confirmed using a recombinant virus generated by reverse genetics. The parental recombinant influenza C virus, which has the consensus sequences of C/Ann Arbor/1/50 (the residue 24 of M1 is alanine), exhibited a filamentous morphology. In contrast, a recombinant mutant virus, rMG96A, which has an Ala→Thr mutation at residue 24 of M1, exhibited spherical morphology. The mutant M1 protein had lower membrane affinity than did the wild type M1, suggesting that the difference in the affinity affects the virion morphology (Muraki et al., 2007). Furthermore, the flotation analysis showed that budding of influenza C virus does not occur from the lipid raft domain of the plasma membrane. These studies indicate that M1 plays an important role in virus budding, and imply that a region around the residue 24 of M1 may represent a late domain of the protein and that budding mechanism of influenza

The fact that the biochemical characteristics of CM2 are closely similar to those of influenza A virus M2 and that CM2 seems to have permeability to proton suggests that CM2 is also crucial in influenza C virus replication. To clarify the role(s) of CM2 in the influenza C virus replication cycle, attempts were made to generate a recombinant CM2-deficient virus using reverse genetics. However, infectious recombinants lacking CM2 have not been rescued to date (Furukawa et al., 2011), which highly suggests that CM2 is essential to influenza C

Using VLP generation system, evidence was obtained that CM2 is involved in the packaging and uncoating processes. The CM2-deficient influenza C VLPs were successfully generated and the amount of GFP-vRNA in the VLPs was quantified. The data showed that the CM2-deficient VLPs contain approximately 37% of the vRNA found in wild-type VLPs, although no significant differences were detected in the expression levels of viral components in VLP-producing cells as well as in the number and morphology of the generated VLPs (Furukawa et al., 2011). This finding suggests that CM2 is involved in the mini-genome packaging into VLPs. In addition, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced GFP expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of GFP-vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner (Furukawa et al., 2011). Thus, CM2 appears to play a role in the packaging and uncoating processes of the virus replication cycle. However, whether and

how the CM2 channel activities relate to the roles remains unclear.

residue 24 of the M1 protein is a determinant for virion morphology.

C virus differs from that of influenza A virus.

**4.2 CM2 protein**

virus replication.

As mentioned above, P42, encoded by a collinear transcript of the RNA segment 6 (M gene), is cleaved by signal peptidase to generate CM2, composed of the C-terminal 115 amino acids, in addition to the M1' protein, composed of the N-terminal 259 amino acids (Hongo S et al., 1999; Pekosz & Lamb 1998). It is of interest to clarify the role(s) of P42 and M1' in virus replication. The characteristics of the P42 and M1' (or p31) proteins were precisely determined as follow.

The M1' protein was found to undergo rapid degradation after cleavage from P42, since addition of the 26S proteasome inhibitor lactacystin to the cells infected with influenza C virus or transfected with M gene cDNA drastically reduced M1' degradation (Pekosz & Lamb 2000). In addition, the hydrophobic nature, not the specific amino acid sequence, of the 17-amino-acid C terminus of M1' was demonstrated to act as the signal for targeting the protein to membranes and for degradation (Pekosz & Lamb 2000). Thus, M1' seems to be dispensable to virus replication.

A series of mutant M gene cDNAs were transfected into COS-1 cells to examine the characteristics of P42 (Li et al., 2004). P44, the N-glycosylated form of P42, forms disulfidelinked dimers and tetramers. P44 is transported to the Golgi apparatus, but not to the trans-Golgi, since P44 is sensitive to endoglycosidase H. P44 and P42 are unstable irrespective of N-glycosylation or oligomerization. The addition of lactacystin prevented the degradation of P42 as well as M1', but not that of P44 efficiently, suggesting that P44 is degraded by another protease besides the 26S proteasome (Li et al., 2004). The role(s) of P42/P44 in virus replication remains to be clarified.

#### **5. Regulation of M1 and CM2 synthesis**

In influenza A virus-infected cells, the regulatory machanism for viral mRNA splicing has been extensively investigated. It has been reported that the steady-state level of spliced viral

Influenza C Virus: Structure and Function of M Gene and Its Products 71

Crescenzo-Chaigne, B. & van der Werf, S. (2001). Nucleotides at the Extremities of the Viral RNA

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Herrler, G. & Klenk, H.D. (1991). Structure and Function of the HEF Glycoprotein of

Homma, M.; Ohyama, S. & Katagiri, S. (1982). Age Distribution of the Antibody to Type C Influenza Virus. *Microbiology and Immunology,* Vol.26, pp.639–642, ISSN 0385-5600 Hongo, S.; Sugawara, K.; Nishimura, H.; Muraki, Y.; Kitame, F. & Nakamura, K. (1994).

Hongo, S.; Sugawara, K.; Muraki, Y.; Kitame, F. & Nakamura, K. (1997). Characterization of

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Hongo, S.; Ishii, K.; Mori, K.; Takashita, E.; Muraki, Y.; Matsuzaki, Y. & Sugawara, K. (2004).

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Shimotai, Y. & Hongo, S. (2011). Role of the CM2 Protein in the Influenza C Virus Replication Cycle. *Journal of Virology,*Vol.85, No.3, pp.1322–1329, ISSN 0022-538X Garaigorta, U. & J. Ortin, J. (2007). Mutation Analysis of a Recombinant NS Replicon Shows

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Identification of a Second Protein Encoded by Influenza C Virus RNA Segment 6.

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Nakamura, K. (1998). Identification of a 374 Amino Acid Protein Encoded by RNA Segment 6 of Influenza C Virus. *Journal of General Virology,* Vol.79, pp.2207-2213,

K. (1999). Influenza C Virus CM2 Protein is Produced From a 374-Amino-Acid Protein (P42) by Signal Peptidase Cleavage. *Journal of Virology,* Vol.73, No.1, pp.46–

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transcripts is only 10% that of unspliced viral transcripts (Palese & Shaw, 2007). The inefficient splicing can be understood partly by the fact that influenza A virus NS1 protein is associated with spliceosomes and inhibits pre-mRNA splicing (Fortes et al., 1994; Garaigorta & Ortin, 2007; Lu et al., 1994). The cis-acting sequences in the NS1 transcript also negatively regulates splicing (Nemeroff et al., 1992). The splicing of influenza A virus M1 mRNA is controlled by the rate of nuclear export (Valcarcel et al., 1993), and the splicing of influenza A virus M1 mRNA is regulated by the binding of the viral polymerase complex and cellular splicing factor SF2/ASF (Shih et al., 1995 ; Shih & Krug, 1996).

The influenza C virus NS1 protein (C/NS1), a product of the RNA segment 7 (NS gene), regulates the splicing efficiency of the M gene (Muraki et al., 2010). In influenza C virusinfected cells, the predominant transcript from the M gene is a spliced mRNA (Hongo et al., 1994). Ribonuclease protection assays showed that the splicing efficiency of the M gene mRNA in infected cells was much higher than that in M gene cDNA-transfected cells, suggesting that viral factor(s) facilitates the M gene mRNA splicing. To investigate the factor(s) involved in the splicing, Muraki et al. analyzed cells co-transfected with M and NS gene-cDNAs, and showed that the splicing of the M gene mRNA was enhanced by expressing C/NS1 (Muraki et al., 2010). The fact was obtained that splicing ratio of influenza A virus M gene mRNA was also increased by C/NS1, suggesting that C/NS1 has an intrinsic ability of up-regulating viral mRNA splicing. Therefore, we speculate that efficient splicing of M gene mRNA leads to a high and reduced level of M1 and CM2 expression, respectively, creating conditions that are optimal for virus replication.

#### **6. Conclusion**

The RNA segment 6 (M gene) of influenza C virus encodes the M1 and CM2 proteins. It has been demonstrated that the M1 protein is a determinant for virion morphology and CM2 is involved in packaging and uncoating processes. A further study will be needed to clarify the structure-function relationship more precicely.

#### **7. Acknowledgements**

The author thanks all the laboratory members of Department of Microbiology, Kanazawa Medical University School of Medicine and Department of Infectious Diseases, Yamagata University Faculty of Medicine for helpful assistance and discussion.

#### **8. References**

transcripts is only 10% that of unspliced viral transcripts (Palese & Shaw, 2007). The inefficient splicing can be understood partly by the fact that influenza A virus NS1 protein is associated with spliceosomes and inhibits pre-mRNA splicing (Fortes et al., 1994; Garaigorta & Ortin, 2007; Lu et al., 1994). The cis-acting sequences in the NS1 transcript also negatively regulates splicing (Nemeroff et al., 1992). The splicing of influenza A virus M1 mRNA is controlled by the rate of nuclear export (Valcarcel et al., 1993), and the splicing of influenza A virus M1 mRNA is regulated by the binding of the viral polymerase complex and cellular

The influenza C virus NS1 protein (C/NS1), a product of the RNA segment 7 (NS gene), regulates the splicing efficiency of the M gene (Muraki et al., 2010). In influenza C virusinfected cells, the predominant transcript from the M gene is a spliced mRNA (Hongo et al., 1994). Ribonuclease protection assays showed that the splicing efficiency of the M gene mRNA in infected cells was much higher than that in M gene cDNA-transfected cells, suggesting that viral factor(s) facilitates the M gene mRNA splicing. To investigate the factor(s) involved in the splicing, Muraki et al. analyzed cells co-transfected with M and NS gene-cDNAs, and showed that the splicing of the M gene mRNA was enhanced by expressing C/NS1 (Muraki et al., 2010). The fact was obtained that splicing ratio of influenza A virus M gene mRNA was also increased by C/NS1, suggesting that C/NS1 has an intrinsic ability of up-regulating viral mRNA splicing. Therefore, we speculate that efficient splicing of M gene mRNA leads to a high and reduced level of M1 and CM2

expression, respectively, creating conditions that are optimal for virus replication.

The RNA segment 6 (M gene) of influenza C virus encodes the M1 and CM2 proteins. It has been demonstrated that the M1 protein is a determinant for virion morphology and CM2 is involved in packaging and uncoating processes. A further study will be needed to clarify the

The author thanks all the laboratory members of Department of Microbiology, Kanazawa Medical University School of Medicine and Department of Infectious Diseases, Yamagata

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**6. Conclusion**

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**5**

Renata Coura

*France*

**Viral Vectors in Neurobiology:**

**Therapeutic and Research Applications**

 *Centre de Neuroscience Paris Sud – CNPS – Université Paris Sud XI*

Viruses are intracellular parasites with simple DNA or RNA genomes (Figure 1A). Three steps compose virus life cycle: infection of a host cell, replication of its genome within the host cell environment, and formation of new virions (Figure 1B). This process is often but

Fig. 1. Virus. (A) Structure. Simplified scheme of virus structure, with a lipid envelope that can be present or not; a protein-composed capsid and the genetic material, that can be DNA or RNA, double or single strand. (B) Life cycle. Example of the course of adeno-associated virus (AAV) productive infection. Scheme showing the eight steps of AAV transduction of host cells: (1) viral binding to a membrane receptor/co-receptor; (2) endocytosis of the virus by the host cell; (3) virus intracellular trafficking through the endosomal compartment; (4) escape of the virus from the endosome; (5) virion uncoating; (6) entry into the nucleus; (7) viral genome conversion from a single-stranded to a double-stranded genome; and (8) integration into the host genome or permanence of an episomal form capable of expressing

**1. Introduction**

**1.1 History and definition of viral vectors**

an encoded gene (from Coura and Nardi, 2008).

## **Viral Vectors in Neurobiology: Therapeutic and Research Applications**

Renata Coura

 *Centre de Neuroscience Paris Sud – CNPS – Université Paris Sud XI France*

#### **1. Introduction**

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#### **1.1 History and definition of viral vectors**

Viruses are intracellular parasites with simple DNA or RNA genomes (Figure 1A). Three steps compose virus life cycle: infection of a host cell, replication of its genome within the host cell environment, and formation of new virions (Figure 1B). This process is often but

Fig. 1. Virus. (A) Structure. Simplified scheme of virus structure, with a lipid envelope that can be present or not; a protein-composed capsid and the genetic material, that can be DNA or RNA, double or single strand. (B) Life cycle. Example of the course of adeno-associated virus (AAV) productive infection. Scheme showing the eight steps of AAV transduction of host cells: (1) viral binding to a membrane receptor/co-receptor; (2) endocytosis of the virus by the host cell; (3) virus intracellular trafficking through the endosomal compartment; (4) escape of the virus from the endosome; (5) virion uncoating; (6) entry into the nucleus; (7) viral genome conversion from a single-stranded to a double-stranded genome; and (8) integration into the host genome or permanence of an episomal form capable of expressing an encoded gene (from Coura and Nardi, 2008).

Viral Vectors in Neurobiology: Therapeutic and Research Applications 77

Fig. 2. Example of vector construction. (A) Wild AAV. (B) AAV recombinant vector production. AAV vector cassette, containing only the ITRs of the wild virus and the

and Nardi, 2008).

transgene and its promoter; helper cassette, containing the AAV rep and cap genes required for virus packaging and the Ad genes E2, VA and E4, required for virus replication. (1); cotransfection of both plasmids in HEK 293 packaging cells, whose genome contains the Ad E1 gene, which together with the other Ad required genes, supplied by the helper plasmid, allows the establishment of a productive infection (2); empty capsid is formed by AAV structural proteins assembly into the nucleolus (3); the replicated ssDNA viral genome with the transgene flanked by ITRs (vector plasmid) are packaged into the empty capsid in the nucleoplasm (4), giving rise to a non-replicative recombinant AAV vector virion (5). The regulatory proteins act especially in the replication and packaging processes (from Coura

not always associated with pathogenic effects against the host organism. Nevertheless, since the mid-1980s, a likely useful role for virus has been envisaged. The idea is to use the unique virus capacity to enter the cell and to replicate their genome to construct vectors, containing the viral envelope and a recombinant genome, so that these vectors could be able to deliver genetic material into cells. Then, recombinant viral vectors are created in which genes essential for viral replication are removed and a gene of interest is inserted in the viral genome (Figure 2). While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Meanwhile, defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral *gene expression* (see **glossary**) within target cells. These viral vectors would be able to long-term gene delivery to mammalian cells, without pathogenicity and with minimal associated toxicity. Today, several viral vector systems are close to achieving this aim, providing stable transgenic expression in many different cell types and tissues.

#### **1.2 Viral vectors**

For the production of an efficacious and safe viral vector it is required at first to identify the crucial viral sequences for viral particle assembly, for viral genome package, and for *transgene* (see **glossary**) delivery to target cells. Then, dispensable genes are deleted from viral genome in order to reduce its pathogenicity and immunogenicity. At last, residual viral genome and transgene are integrated into the construct (Figure 2). Some viral vectors are able to integrate host genome unlike others that remain in an episomal form. Integrative vectors, like retroviruses and adeno-associated vectors are able to promote a persistent transgene expression. Otherwise, non-integrative vectors, like adenovirus whose viral DNA is maintained in an episomal form into infected cells, lead to a transient transgene expression. Each vector presents specific advantages and limitations that become more or less proper depending on the objective of its application (http://cmbi.bjmu.edu.cn/cmbidata/therapy/research/re02/021.htm; Osten et al., 2007; Howarth et al., 2010).

The ideal vector has not been described yet, but its characteristics should include:

Most vectors used for gene delivery are derived from human viral pathogens that have been made nonpathogenic by deleting essential viral genes. They usually have a broad tropism; therefore they can infect and deliver their encoded transgenes to a wide spectrum of cells and/or tissues.

Currently, the most efficient and commonly used viral vectors are adenovirus (Ad), adenoassociated virus (AAV), herpes simplex virus type 1-derived vectors (HSV-1), and retrovirus/lentivirus vectors (Table 1).

not always associated with pathogenic effects against the host organism. Nevertheless, since the mid-1980s, a likely useful role for virus has been envisaged. The idea is to use the unique virus capacity to enter the cell and to replicate their genome to construct vectors, containing the viral envelope and a recombinant genome, so that these vectors could be able to deliver genetic material into cells. Then, recombinant viral vectors are created in which genes essential for viral replication are removed and a gene of interest is inserted in the viral genome (Figure 2). While this eliminates pathogenicity due to viral replication, retention of viral genes and continued expression of these genes may limit the potential of the current generation of vectors. Meanwhile, defective viral vectors represent a different approach, in which only viral recognition signals are used to allow packaging of foreign DNA into a viral coat while eliminating the possibility of viral *gene expression* (see **glossary**) within target cells. These viral vectors would be able to long-term gene delivery to mammalian cells, without pathogenicity and with minimal associated toxicity. Today, several viral vector systems are close to achieving this aim, providing stable transgenic expression in many

For the production of an efficacious and safe viral vector it is required at first to identify the crucial viral sequences for viral particle assembly, for viral genome package, and for *transgene* (see **glossary**) delivery to target cells. Then, dispensable genes are deleted from viral genome in order to reduce its pathogenicity and immunogenicity. At last, residual viral genome and transgene are integrated into the construct (Figure 2). Some viral vectors are able to integrate host genome unlike others that remain in an episomal form. Integrative vectors, like retroviruses and adeno-associated vectors are able to promote a persistent transgene expression. Otherwise, non-integrative vectors, like adenovirus whose viral DNA is maintained in an episomal form into infected cells, lead to a transient transgene expression. Each vector presents specific advantages and limitations that become more or less proper depending on the objective of its application (http://cmbi.bjmu.edu.cn/cmbidata/therapy/research/re02/021.htm; Osten et al., 2007;

The ideal vector has not been described yet, but its characteristics should include:

Absence of toxicity to target cells and undesirable effects like as immune response

Capacity of site-specific integration, allowing a long-term transgene expression, as in

Most vectors used for gene delivery are derived from human viral pathogens that have been made nonpathogenic by deleting essential viral genes. They usually have a broad tropism; therefore they can infect and deliver their encoded transgenes to a wide spectrum of cells

Currently, the most efficient and commonly used viral vectors are adenovirus (Ad), adenoassociated virus (AAV), herpes simplex virus type 1-derived vectors (HSV-1), and

Easy and efficient production of high titers of viral particle;

against the vector or the transgene;

 Capacity of transduction of specific cell types; Infection of proliferative and quiescent cells.

cases of genetic disorders;

retrovirus/lentivirus vectors (Table 1).

different cell types and tissues.

**1.2 Viral vectors**

Howarth et al., 2010).

and/or tissues.

Fig. 2. Example of vector construction. (A) Wild AAV. (B) AAV recombinant vector production. AAV vector cassette, containing only the ITRs of the wild virus and the transgene and its promoter; helper cassette, containing the AAV rep and cap genes required for virus packaging and the Ad genes E2, VA and E4, required for virus replication. (1); cotransfection of both plasmids in HEK 293 packaging cells, whose genome contains the Ad E1 gene, which together with the other Ad required genes, supplied by the helper plasmid, allows the establishment of a productive infection (2); empty capsid is formed by AAV structural proteins assembly into the nucleolus (3); the replicated ssDNA viral genome with the transgene flanked by ITRs (vector plasmid) are packaged into the empty capsid in the nucleoplasm (4), giving rise to a non-replicative recombinant AAV vector virion (5). The regulatory proteins act especially in the replication and packaging processes (from Coura and Nardi, 2008).

**Herpes Simplex Virus (HSV)**   Icosahedric Enveloped

Kb

fusion Episomal (nonintegrative) Capable of transducing postmitotic cells Persistence of

Toxicity

**1.2.1 Adenovirus**

disintegration.

deficient.

Genome capacity of 30

Entry into the cell by

expression for weeks Difficult production

Table 1. Main viral vectors systems and their characteristics.

Viral Vectors in Neurobiology: Therapeutic and Research Applications 79

Adenoviruses (Ads) are medium-sized (90–100 nm), non-enveloped icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. The viral genome is large, consisting of a single double-stranded DNA molecule 36 to 38 kilobases in size. Viral DNA replication and transcription are complexes, and viral replication and assembly occur only in the nucleus of infected cells. Mature virions are released by cellular

There are 55 described serotypes in humans, which are responsible for 5–10% of upper

Adenoviral infection is initiated by the virus binding to the cellular receptor. Internalization occurs via receptor-mediated endocytosis followed by release from the endosome. After endosomal release, the viral capsid undergoes disassembly while moving to the nuclear pore. Nuclear entry of the viral DNA is completed upon capsid dissociation, and the viral

Since adenoviral replication depends on the E1A region of the viral genome, all recombinant adenoviral vectors have this region of its genome deleted, and are referred to as "replicationdeficient" virus. Such vectors are capable of infecting a cell only once, no viral propagation occurs, and the infected cell does not die as a result of vector infection. As replicationdeficient viruses are required, the 293 cell line is utilized for vector production. This is a human kidney cell line which has been stably transfected with the E1A region of the adenoviral genome. This allows the vector to be made and matured within the 293 cell, yet vectors prepared from this cell line will lack the E1A region and remain replication-

The non-integrative feature of adenovirus limits their use in basic research, although adenoviral vectors are occasionally used in *in vitro* experiments. Their primary applications are in gene therapy, especially oncolytic gene therapy, and vaccination (Ayuso et al., 2010).

respiratory infections in children, and many infections in adults as well.

DNA does not integrate into the host genome but remains in an episomal state.

 System currently under development Current vectors provide transient expression Low transduction efficiency Toxicity

 Neuronal gene transfer, *in vitro* and *in*

*vivo*.

 Large insert size Could provide long- term CNS gene expression High titer

Table 1. Main viral vectors systems and their characteristics.

#### **1.2.1 Adenovirus**

78 Molecular Virology

High

 Transient expression Viral proteins can be expressed in host following vector administration

 Insertional mutagenesis (random integration) Low titers (106- 107) *In vivo* delivery remains poor. Effective only when infecting helper cell

lines

Limited capacity for DNA fragments insertion

immunogenicity, inducing an important cellular and humoral immune response that can be fatal;

Therapies that require transient gene expression: cancer therapy, angiogenesis induction and DNA vaccine production (due

to its inflammatory

and

immunogenic properties);

 Genetic diseases of T cells and hematological diseases (Retrovirus); HIV/AIDS Optogenetics Neuronal tracing

 Genetic Diseases Tumors Neurological Diseases Ocular Diseases Cardiovascular Diseases Others

**Virus vector Description Advantages Limitations Applications**

 Easy propagation in high titers(1010-

 High transduction efficiency; Infection of the majority of cell types; Possibility of insertion of large DNA fragments (up to 8kb); Infects both replicating and differentiated cells.

1013);

Low

Kb);

host

immunogenicity; Easy propagation in high titers; Infection of the majority of cell types; Long-term gene expression

Low

immunogenicity; Possibility of insertion of modest DNA fragments (up to 8

 High transduction efficiency; Integrates into host genome resulting in sustained expression of vector Extremely well studied system Vector proteins not expressed in

**Adenovirus (Ad)**

**Retrovirus (Retrovirus and Lentivirus)**

**Adenoassociated virus (AAV)**

 Icosahedric Non- enveloped Genome capacity of 36

 Entry into the cell by receptor binding Episomal (nonintegrative) Capable of transducing postmitotic cells Persistence of

expression for weeks

Genome capacity of 8

Entry into the cell by

or months Difficult production

Toxicity

 Globular Enveloped

Kb

fusion Integrative Capable of transducing proliferative (retrovirus and lentivirus) and quiescent (lentivirus)

cells Persistence of expression for years Easy production Minimal toxicity

 Icosahedric Non- enveloped Genome capacity of

4.7 Kb

No toxicity

 Entry into the cell by receptor binding Integrative Capable of transducing postmitotic cells Persistence of expression for years Difficult production

Kb

Adenoviruses (Ads) are medium-sized (90–100 nm), non-enveloped icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. The viral genome is large, consisting of a single double-stranded DNA molecule 36 to 38 kilobases in size. Viral DNA replication and transcription are complexes, and viral replication and assembly occur only in the nucleus of infected cells. Mature virions are released by cellular disintegration.

There are 55 described serotypes in humans, which are responsible for 5–10% of upper respiratory infections in children, and many infections in adults as well.

Adenoviral infection is initiated by the virus binding to the cellular receptor. Internalization occurs via receptor-mediated endocytosis followed by release from the endosome. After endosomal release, the viral capsid undergoes disassembly while moving to the nuclear pore. Nuclear entry of the viral DNA is completed upon capsid dissociation, and the viral DNA does not integrate into the host genome but remains in an episomal state.

Since adenoviral replication depends on the E1A region of the viral genome, all recombinant adenoviral vectors have this region of its genome deleted, and are referred to as "replicationdeficient" virus. Such vectors are capable of infecting a cell only once, no viral propagation occurs, and the infected cell does not die as a result of vector infection. As replicationdeficient viruses are required, the 293 cell line is utilized for vector production. This is a human kidney cell line which has been stably transfected with the E1A region of the adenoviral genome. This allows the vector to be made and matured within the 293 cell, yet vectors prepared from this cell line will lack the E1A region and remain replicationdeficient.

The non-integrative feature of adenovirus limits their use in basic research, although adenoviral vectors are occasionally used in *in vitro* experiments. Their primary applications are in gene therapy, especially oncolytic gene therapy, and vaccination (Ayuso et al., 2010).

Viral Vectors in Neurobiology: Therapeutic and Research Applications 81

stranded DNA. AAV is one of the smallest viruses with a capsid of approximately 22 nm and one of the most spread of this family, leading to seropositivity in more than 80% of human population (serotype 2). Despite this high seroprevalence, the virus has not been linked to any human illness, causing a very mild immune response. Because a co-infecting helper virus is usually required for a productive infection to occur, AAV serotypes are

The wild AAV has a linear single-stranded DNA genome of approximately 4.7 Kb of either plus (sense) or minus (anti-sense) polarity. The AAV2 DNA termini consist of a 145 nucleotide-long inverted terminal repeat (ITR) that forms a characteristic T-shaped hairpin structure. These ITRs are important in the site-specific integration of AAV DNA into a specific site in chromosome 19. The ability of wild-type AAV to selectively integrate into chromosome 19 made them an attractive candidate for the production of a gene therapy vector that could do the same. The ITRs act as DNA replication origin, as well as signal for package and integration. In addition, they also act as regulator element for wild AAV gene expression. The ITRs flank the two viral genes rep (replication) and cap (capsid) encoding nonstructural and structural proteins, respectively. The virus does not encode a polymerase

ascribed to a separate genus in the Parvoviridae family designated Dependovirus.

relying instead on cellular polymerase activity to replicate its DNA (Ni et al., 1998).

removed by either density gradient centrifugation or heat inactivation.

Nardi, 2008; Mezzina & Merten, 2011).

**1.2.4 Herpes simplex virus**

Adeno-associated vectors are prepared by replacing the capsid genes with the gene of interest. Construction of AAV vectors consists of the recombinant AAV vector plasmid DNA, and a non-rescuable AAV helper plasmid, which encodes for the AAV capsid proteins. Also required is either wild-type adenovirus or HSV and cell line for viral propagation. Unlike the previous vector systems described, the cell line need not contain any portion of the AAV genome since all required AAV genome elements could be provided by the two plasmids. Cells are first infected with the wild-type adenovirus or HSV, and then both the recombinant AAV vector plasmid DNA and the non-rescuable AAV helper plasmid are co-transfected into the cells. The cells produce mature recombinant AAV vectors as well as wild-type adenovirus or HSV. The wild-type adenovirus or HSV is

These vectors have been designed to produce a gene therapy vector with site-specific integration and the ability to infect multiple cell types. Unfortunately, this has not been the case to date for these vectors. Current research focuses on how to regain the site-specific integration sequences into the recombinant vector. These vectors do offer some advantages over other vector systems which include the lack of initiating an immune response, their stability and ability to infect a variety of dividing and non-dividing cells. Unfortunately, they cannot incorporate genes larger than 5 kb and must be closely screened for adenoviral or HSV contamination (Berns, 1996; Grimm & Kay, 2003; Coura & Nardi, 2007; Coura &

Herpes simplex viruses are members of the herpes virus family, Herpesviridae, that infect humans. They are ubiquitous neurotropic and neuroinvasive viruses that persist in the body by becoming latent and hiding from the immune system in the cell bodies of nerves. Latency is defined as a state in which viral DNA is maintained within the cell nucleus in the absence of any viral replication. The structure of herpes viruses consists of a relatively large double-

#### **1.2.2 Retrovirus**