Structure and life cycle - Influenza A viruses

1. Influenza A viruses

1.3 Structure and life cycle

Influenza A viruses (Fig.1) have a spherical shape with a diameter of 80-120 nm, they are enveloped with a lipid membrane derived from the plasma membrane of the infected cell. There are three proteins, HA, NA, and matrix 2 protein (M2, ion channel protein), embedded in this membrane. Below the lipid membrane is the matrix protein 1 (M1) which determines the shape of influenza A viruses and interacts with the cytoplasmic domains of HA, NA and M2 proteins, and thus mediates a connection with the ribonucleoproteins (RNPs). The genome of influenza A viruses is composed of eight segments of single-stranded, negative-sense RNA, about 13 kb in length, and encodes at least 13 proteins including HA, NA, M1, M2, nucleoprotein (NP), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), PB1-F2 protein, nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), also named as nuclear export protein (NEP), as well as two new proteins PA-X and PB1-N40 which are synthesized from PA mRNA and PB1 mRNA, respectively (Bouvier and Palese, 2008; Chen et al.,2001; Muramoto et al, 2013;

Vasin et al., 2014). Among these proteins, HA, NA, M1, M2, NP, PA, PB1 and PB2 are structural proteins, NS1, NS2 (NEP), PB1-F2, PA-X and PB1-40 are accessory proteins (Goraya et al., 2015; Jagger et al., 2012).

Fig. 1 Schematic diagram of an influenza A virus (Wendel et al., 2015)

The life cycle (Fig. 2) of influenza A viruses is initiated by the interaction of the HA protein and cellular receptors on the surface of target cells. During this process, HA mediates the binding to its receptor, and the fusion of viral and cellular membranes which results in viral ribonucleoproteins (vRNPs) released into the cytoplasm. Then vRNPs are transported into the nucleus where the replication of the viral genome and the transcription of messenger RNAs (mRNA) is carried out by PA, PB1, PB2, NP and PB1-F2 proteins. Proteins required for transcription and replication are translated after mRNAs have been transported to the cytoplasm and imported back to the nucleus. The M1 protein and NEP protein export newly synthesized vRNPs from the nucleus to the cytoplasm. Virions are assembled at the plasma membrane and released from infected cells with the help of the NA protein which releases sialic acids and thus inactivates virus receptors on the cell surface.

Fig. 2 Schematic diagram of the influenza viral life cycle (Zheng et al., 2013) 1.4 Proteins

1.4.1 Hemagglutinin

The HA protein is an important protein in the viral membrane, encoded by segment 4 and folded as a rod-shaped trimer. HA protein occurs in 18 subtypes. H17 and H18 subtypes are exceptional as they belong to bat viruses; other subtypes are divided into

two groups on the basis of the sequence and structural properties. H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 subtypes belong to group 1, group 2 consists of H3, H4, H7, H10, H14 and H15 subtypes (Russell et al., 2004). The HA protein determines the host tropism of influenza A viruses by interaction with virus receptors (sialic acid-containing macromolecules) expressed on the cellular surface of target cells.

(Chen et al., 1998; Goraya et al., 2015; Lamb and Choppin, 1983; Skehel and Wiley, 2000; Wilson et al., 1981). The HAs of human and swine influenza A viruses preferentially bind to α-2,6-linked sialic acid, whereas avian influenza A viruses have a preference to recognize α-2,3-linked sialic acid. This property is consistent with α-2,6-linked sialic acid being predominant on epithelial cells in the human and swine airways and α-2,3-linked sialic acid being present on epithelial cells in the intestinal tract of birds (Ito et al., 1998). The binding specificity of influenza A viruses is determined by some domains within the receptor binding site of HA: the 130-loop, the 220-loop and the 190-α helix (Weis et al., 1988; Wilson et al., 1981). Some mutations of amino acid residues in these domains may result in the change of the binding specificity. For example, Gln and Gly residues at position 226 and 228 of H2 and H3 HA subtypes are associated with a preferential binding to α-2,3-linked sialic acid, whereas Leu and Ser residues at these two positions determine a preference for α-2,6-linked sialic acid. For the H1 subtype, Asp residues at position 190 and 225 confer preferential binding to α-2,6-linked sialic acid, whereas Glu and Gly at these positions recognize α-2,3-linked sialic acid preferentially (Steven et al., 2004).

Furthermore, the receptor binding site of HA protein has antigenic properties and can induce the infected host to generate protective antibodies against the corresponding virus subtype by blocking virus attachment (Mallajosyula et al., 2014).

In addition to binding to the receptors and determining the binding specificity and affinity, HA mediates the fusion between the viral and cellular membranes to release viral RNP into infected cells. This fusion function is performed by the HA2 polypeptide which is activated after HA cleavage by proteases. Under low pH-conditions, the cleaved HA undergoes an irreversible change which brings the N-terminus of the activated HA2 polypeptide into a position to mediate the fusion of viral and cellular membrane (Carr and Kim, 1993; Skehel and wiley, 2000; Weber et

al., 1994; Wharton et al., 1995). The cleavage efficiency is determined by the available proteases and the amino acid sequence of the cleavage site. In this way, HA plays a crucial role in the viral virulence, tropism and pathogenicity (Garten and Klenk, 1999; Skehel and wiley, 2000). Highly pathogenic influenza viruses have a polybasic sequence R-X-R/K-R at the cleavage site of HA. This sequence can be recognized specifically and cleaved efficiently by ubiquitous proteases (furin-like enzymes), which are distributed in a wide range of tissues, resulting in systemic infection and systemic symptoms (Bottcher-Friebertsgauser et al., 2013; Suguitan et al., 2012). In contrast, low-pathogenic influenza viruses have a monobasic sequence (single Arg residue) Q/E-X-R at the cleavage site of HA. This sequence can be recognized and cleaved efficiently by trypsin-like enzymes, which are present on epithelial cells of the respiratory tract and/or intestinal tract, and lead to local infections. A change in the cleavage site and thus in the cleavability of HA is crucial for the change of influenza A viruses from low-virulence to highly virulent viruses (Horimoto and Kawaoka, 2005; Hirst et al., 2004).

1.4.2 Neuraminidase

The NA protein is encoded by segment 6 and folded as a mushroom-shaped tetramer.

Each monomer is composed of three domains: a membrane anchor, a rod-shaped stalk region, and a globular head which possesses the enzyme site, respectively (Air, 2012;

Gamblin and Skehel, 2010). The catalytic site within the globular head domain is lined by some conserved amino acids: Arg118, Asp151 and others (Colman et al., 1989). The globular head domain includes not only the enzyme site, but also a calcium binding site (Bossart-whitaker et al., 1993; Tulip et al., 199). Except N10 and N11 subtypes discovered recently in bats (Tong et al., 2012), N1-N9 subtypes of influenza A viruses NA are divided into two groups. N1, N4, N5 and N8 subtypes belong to group 1, and group 2 includes N2, N3, N6, N7 and N9 subtypes. On the viral surface, the NA protein is surrounded by the most predominant protein HA (Harris et al., 2006). During the viral life cycle, NA protein contributes three function:

(i) helping the virus to approach the epithelial cell surface of the respiratory tract by desialylation of inhibitory mucins, (ii) facilitating the release of newly synthesized virions from infected cells by releasing sialic acid from cell surface receptors and (iii)

preventing the aggregation of viral particles by removing sialic acids from the viral surface (Matrosovish et al., 2004; Wagner et al., 2000; Wagner et al., 2002). The amount of NA protein on the viral surface is much lower compared to that of the HA protein, but antigenic epitopes surrounding the enzyme site within the globular head domain can trigger the host to produce antibodies that prevent influenza A virus infections (Air, 2012; Air et al., 1990).

1.4.3 Matrix protein

Matrix protein M1 of influenza A virus together with the M2 protein are encoded by segment 7 (Horimoto and Kawaoka, 2001; Webster et al., 1992). These two proteins are important for assembly and morphology of influenza A virus (Liu et al., 2017).

The M1 protein is the most abundant protein in the viral particle, and forms M1-M1 interphases at the inner side of the membrane which determine the viral structure at different stages of the life cycle depending on the pH value and which affect the viral budding (Harris et al., 2001; Safo et al., 2014). M1 protein has 252 amino acid residues and comprises three domains: the N-terminal domain, the middle domain and the C-terminal domain. The M1 protein not only interacts with HA and NA, but also binds to newly synthesized RNP to transport vRNPs from the nucleus to the cytoplasm with the help of NS2 for packaging in new virions (Ali et al., 2000; Cao et al., 2012; Jin et al., 1997; Noton et al., 2007; Ye et al., 1999; Zhang et al., 200). M2 protein has 97 amino acid residues folded as tetramers and is the third protein on the viral surface. As an ion channel, M2 is crucial for the viral RNP release into the cytoplasm by eliciting viral uncoating during the viral entry process (Edinger et al., 2014). In addition to the effect on viral morphology and infectivity, M2 protein also affects the interaction between NP and RNA during virus assembly (Chen et al., 2008;

Iwatsuki-Horimoto et al., 2006; McCown and Pekosz, 2005; McCown and Pekosz, 2006; Rossman et al., 2010).

1.4.4 Non-structural protein

Non-structural proteins of influenza A virus include two proteins: NS1 and NS2 that are encoded by segment 8 and translated by viral mRNA generated by splicing. The NS1 proteins are expressed in the nucleus and cytoplasm of infected cells, possess an amino acid length ranging from 215 to 237 and include three domains: an N-terminal

RNA-binding domain, a central effector domain and a C-terminal region (Cheng et al., 2018; Hale et al., 2008; Krug, 2015). Based on these domains, NS1 protein contribute several functions during the life cycle of influenza A viruses: they (i) inhibit the transcription and translation of host mRNA, (ii) counteract the innate immunity response (antagonist to type I interferon of host) and (iii) promote the translation of viral mRNA (Fortis et al., 1994; Gack et al., 2009; Garcia-Sastre, 2001; Guo et al., 2007).

The NS2 (NEP) protein is a polypeptide comprising 121 amino acids (Lam and Lai, 1980). At the beginning, this small protein was named as nonstructural protein because it was thought to have no structural function. Subsequently, this protein was found to be present in virions interacting with M1, and demonstrated to have the function to export vRNPs from the nucleus to the cytoplasm in infected cells, to guarantee the packaging of viral genome into new virions on the cellular surface (O'Neill et al., 1998; Richardson and Akkina, 1991; Ward et al., 1995; Yasuda et al., 1993;). Therefore, this protein was renamed as NEP. Apart from export of vRNPs from the nucleus to the cytoplasm, NS2 protein has been shown to possess additional functions during the life cycle of influenza A virus including the contribution to viral budding and the accumulation of viral RNA resulting in the generation of RNA during the early stage of virus replication (Gorai et al., 2012; Mänz et al., 2012; Robb et al., 2009).

1.4.5 Nucleoprotein

NP of influenza A virus is encoded by segment 5. This protein is very conserved (about 95% sequence conservation at protein level) among influenza A viruses (Babar et al., 2015; Reid et al., 2004; Shu et al., 1993; Uyeki, 2003; Vemula et al.,2016; Yang et al., 2008), and abundant in virions. So NP has been used to develop methods for detection of influenza A viruses and for vaccine development against viral infection (Phuong et al., 2018; Zheng et al., 2014). NP protein includes two functional signals:

a nuclear localization signal (NLS) and a nuclear export signal (NES) (Bullido et al., Kudo et al., 1998; Wang et al., 1997; Weber et al., 1998; 2000; Yu et al., 2012) that play an important role in the transport of ribonucleoproteins (RNP), and interact with PB1 and PB2 directly (Biswas et al., 1998; Coloma et al., 2009; Medcalf et al., 1999;

Poole et al., 2004). NP is a multifunctional protein, which not only encapsidates the viral genome to form viral (vRNP) complexes together with PB1, PB2 and PA (Eisfeld et al., 2015), and modulates the replication and transcription of viral RNAs (Eisfeld et al., 2015; Jackson et al., 1982; Herz et al., 1981; Vreede et al., 2011), but also transports vRNP between nucleus and cytoplasm and helps to package the viral genome into new virions (Avalos et al., 1997; Digard et al., 1999; Martin and Helenius, 1991; O’Neill et al., 1995). In order to perform these functions, NP needs to interact with a wide range of factors of virus and infected cells (Eisfeld et al., 2015;

Wang et al., 2017; Watanabe et al., 2014). Furthermore, NP is also needed for the production of messenger RNA, viral genome RNA, and complementary positive-sense RNA in the viral life cycle.

1.4.6 Polymerase proteins and PB1-F2

Polymerase proteins of influenza A virus are crucial components in vRNPs, responsible for transcription and translation of viral RNA, and responsible for mutations in the viral genome during the viral life cycle for better adaptation to new hosts (Miotto et al., 2010; Neumann et al., 2004; Resa-Infante et al., 2011). The 2005; Maier et al., 2008; Sanz-Ezquerro et al., 1995; Yuan et al., 2009). PB1 contains domains which have the ability to bind the promoter of viral RNA and complimentary RNA within the vRNP and complimentary RNP, and play an important role in the RNA synthesis and in the assembly of the polymerase complex (González and Ortín J, 1999; González and Ortín J, 1999; Honda and Ishihama, 1997; Li et al., 1998).PB2 contains two separate sequences providing an RNA cap-binding site, which take caps from host pre-mRNAs in infected cells and initiate viral transcription (Fechter et al., 2003; Guilligay et al., 2008; Honda et al., 1999).

PB1-F2 is a second protein encoded by segment 2, and expressed from +1 within the open reading frame of PB1 gene (Chen et al., 2001). Almost all human isolates of

influenza A virus code for PB1-F2, many animals isolates do not encode this protein.

The full length PB1-F2 encoded by most isolates has 87 or 90 amino acids (Chen et al., 2001; Chen et al., 2004). The length of PB1-F2 in swine influenza viruses is different, for example, classical swine viruses code for a PB1-F2 with 8-11 amino acids, whereas Eurasian avian-like swine viruses code for a polypeptide of 87-89 amino acids (Neumann et al., 2009). This protein can induce apoptosis of infected cells via the by mitochondrial pathway, affect viral replication by interacting with PB1 protein, and affect the viral pathogenicity in mice (Chen et al., 2001; Conenello et al., 2007; Mazur et al., 2008; Zamarin et al., 2005).

2. Genetic variation of influenza A viruses

Mutations and reassortment events result in the generation of new strains and variants of influenza A viruses, which confer the ability to escape the pre-existing host immunity, to change pathogenicity and tissue tropism, and to overcome the species barrier to infect new hosts (Neumann et al., 2009). These new strains and variants are responsible for the yearly epidemics and occasional pandemics. That’s the reason why we have to update influenza vaccines to prevent the threat from influenza A viruses and reduce the economic losses though hosts infected by one subtype of influenza A virus can acquire lifelong immunity to the corresponding strain (Finkenstadt et al.,

Mutations occur in the viral life cycle during the viral RNA replication in a different manner: substitutions (almost 1 × 10^-3 to 8 × 10^-3 sites/year) (Chen and Holmes, 2006), insertions and deletions, due to the viral polymerase which is responsible for replication and transcription of viral RNP but does not possess a proof-reading function (Aggarwal et al., 2010; Koelle et al., 2006; Nobusawa and Sato, 2006; Parvin et al., 1986; Smith et al., 2004). Mutations occurring in glycoproteins may change the viral antigenicity with the result that the pre-existing host immunity cannot prevent infection by these new strains. Several studies have shown that a single mutation occurring in an antigenic site of the H3 subtype may result in the change of antigenicity that enables mutant strains to evade the pre-existing immunity (Hensley et al., 2009; Horimoto and Kawaoka, 2001; Wiley et al., 1981). Mutations causing the characteristic changes of influenza A virus glycoproteins (HA and NA) are designated as antigen drift. In addition to antigenic changes, mutations may also cause a change in the pathogenicity and tropism of the virus. For example, the amino acid at position 627 of PB2 or 66 of PB1-F2 determine the pathogenicity of the H5N1 subtype in mice (Conenello et al., 2007; Hatta et al., 2001); with respect to the position 222 of the HA protein, amino acid D at this position confers the ability to preferentially grow in the

lung, whereas a G at this position enables to virus to grow efficiently in the trachea (Seidel et al., 2014). Furthermore, point mutations are crucial for the viruses in order to adapt to a new host; point mutations occur to survive in the new environment, and to make viral strains more stable (Castelán-Vega et al., 2014; Yang et al., 2017).

2.2 Reassortment events

The genome of influenza A viruses is composed of eight RNA segments, which provide the possibility of reassortment to generate new strains when different subtypes of influenza A viruses co-infect one host (Webster et al., 1982). In this way, an influenza virus may acquire gene segments from another virus, e.g. a human virus may acquire one or more genomic RNA segments from a swine virus. If a human virus acquires the genome segments coding for the viral glycoproteins HA and/or NA from an animal virus, the reassortant virus has antigenic properties that are completely different from those of the human parental virus. Such an event is designated as antigenic shift. Reassortment of influenza A viruses not only occurs in nature but also under laboratory conditions (Kreibich et al., 2013; Neumann et al., 2009). The events play an important role in the evolution of influenza A virus and may be associated with a change of pathogenicity and the switch of host (Dugan et al., 2008; Garten et al., 2009; Holmes et al., 2005; Scholtissek et al., 1978). In history, there were several pandemics in the human population that were caused by reassortant viruses. The most severe pandemic emerged in 1918 and was named Spanish flu. It was caused by an H1N1 virus that may have been transmitted directly from an avian host to humans (Horimoto and Kawaoka, 2005). The latest pandemic emerged in 2009. The genome of the viruses was derived from several subtypes of influenza A viruses. While the reassortment events of the triple reassortant precursor virus have occurred in swine, it is at present not known whether the final reassortment event has occurred in swine or humans (Neumann et al., 2009). Point mutations and reassortment events are the genetic basis for the generation of new virus strains and thus play an important role in the evolution of influenza viruses (Holmes et al., 2005; Nelson and Holmes, 2007).

3. Pigs and swine influenza A viruses 3.1 Pigs

Pigs are susceptible to infection by human and avian influenza A viruses. So genetic reassortment events can occur when a pig is infected by viruses of different subtypes and the two viruses infect the same cell in the airway epithelium (Scholtissek, 1990).

In this way, reassortant viruses have emerged in different parts of the world the genome of which is derived from human/swine influenza A viruses, human/avian influenza A viruses, or human/swine/avian influenza A viruses isolated from pigs in America and other countries (Olsen, 2002; Reperant et al, 2009). Therefore, pigs have been considered as “mixing vessel” for the generation of reassortant viruses (Ito et al., 1998; Ma et al., 2008). Because there is no pre-existing immunity against such new viruses, some of them can overcome species barriers to infect humans or animals of other species, and cause disease or pandemics. Therefore, pigs are considered to play an important role in the interspecies transmission (Horimoto and Kawaoka, 2001;

Imai and Kawaoka, 2012; Kida et al., 1994; Tanner et al., 2015). Previously, it has been tried to explain the role of pigs as mixing vessel for the generation of new influenza variants by the receptor distribution on the airway epithelium. It has been claimed that on the respiratory epithelium of pigs, there are both α-2,6-linked sialic acids and α-2,3-linked sialic acids, i.e receptor determinants for both human and avian influenza viruses. However, careful analysis has shown that the distribution of sialic acids in the human and porcine airways is very similar: α-2,6-linked sialic acids are most abundant in the upper airways; an increasing amount of α-2,3-linked sialic acids is detectable in the lower airways. Because of the similarity of the sialic acid distribution in humans and pigs, there have to be other factors to explain the role of

Im Dokument Use of ex vivo and in vitro cultures of differentiated airway epithelial cells to analyze the infection by influenza A viruses (Seite 15-0)