M. Galdiero* * University of Naples Federico II, Department of Experimental Medicine, Naples, Italy ** University of Naples Federico II, Department of Pharmacy, Naples, Italy † Second University of Naples, Internal Medicine of Clinic Hospital of Marcianise, Department of Medicine, Surgery, Neurology, Geriatric and Metabolic Diseases, ASL Caserta, Italy
The viral family Orthomyxoviridae comprises five genera, but only two are of medical relevance in humans, namely: influenza A and B viruses. Influenza A viruses are further divided into subtypes, based on the antigenic properties of the surface glycoproteins hemagglutinin and neuraminidase. Influenza A virus is a single-stranded, enveloped RNA virus with 8 RNA segments. The initial target of Influenza A infection is the respiratory epithelium and the virus life cycle is initiated by the binding of viral HA to receptors on host cells, which triggers endocytosis of the virus. Once internalized, viral ribonucleoprotein complexes are released into the cytoplasm, transported to the nucleus, where viral replication occurs through the synthesis of new viral components which are transported to the plasma membrane, where the new viruses are formed and exit the infected cell. On average, influenza viruses infect 5–10% of human populations each year, but in some geographical areas or age groups these percentages can heavily increase, especially when a novel influenza virus finds a human population that lacks a suitable neutralizing immune response. We do not yet have an entire understanding of the elements and mechanisms that are fundamental for allowing influenza virus transmission, pandemics generation, and establishment of novel lineages. Influenza virus evolution and consequent pandemics is coarsely driven by two mechanisms: mutations in the viral genome, and reassortment, which is the rearrangement of the eight influenza A viral RNA segments in cells infected with at least two different viruses. Currently, infections due to influenza A virus are responsible for considerable morbidity and mortality worldwide and the loss of life and economic costs are awesome during annual influenza virus epidemics, and can be frightening during worldwide outbreaks of novel strains (pandemics). The present chapter will discuss molecular biology of influenza A viruses together with influenza infections clinical features and therapeutic options.
Influenza is an ancient and deadly disease which has sickened and killed millions of people in local epidemics and global pandemics. Nowadays, it is common knowledge that influenza is a highly infectious viral illness, but before the discovery of viruses the etiological factor of influenza was not known and, therefore, we had to relay solely on the clinical picture characterized by a sudden onset of high fever, cough, headache, muscle and joint pain, unwell feeling, sore throat, and runny nose. These symptoms were clearly described by Hippocrates roughly 2400 years ago, but historical data on influenza were of difficult interpretation, since these symptoms can be similar to those of other respiratory diseases, therefore not distinctive enough.
The word Influenza originated in the 15th century from the Italian language, meaning “influence” since the disease was ascribed to unfavorable astrological influences. A different origin could be the word “influsso” for describing the sweating characteristic of the illness or meaning “influence of the cold.” It was not until 1703 when J. Hugger’s thesis submitted at the University of Edinburgh and named “De Catarrho epidemio, vel Influenza, prout in India occidentali sese ostendit” that the English-spoken world directly associated “influenza” with the disease and its symptoms. After that the name influenza and its shorthand “flu” came into more general use.1
The influenza virus was first isolated from pigs in 1930 by Shope and Lewis.2 This seminal discovery was followed by the isolation in ferrets of influenza A virus by Smith, Andrewes, and Laidlaw.3 In 1936, Burnet demonstrated that influenza virus could be grown in chicken embryonated eggs,4 opening the path for the study of the characteristics of the virus.
It is estimated that influenza virus infects every year 5–10% of the adult population worldwide and 20–30% of the children. Even though most patients recover from flu symptoms within a short period and without serious sequelae, the estimates indicate from 3–5 million cases of serious illness and over 250,000 deaths per year. Therefore, due to its medical importance, influenza viruses have been the focus of extensive research to decipher the molecular mechanisms that dominate cell invasion and pathogenesis.
Influenza virus belongs to the Orthomyxoviridae family, represented by negative-strand RNA viruses whose genome is divided into six to eight individual RNA segments. The orthomyxovirus family name is derived from the Greek words “orthos” which means “correct” while “myxa” stands for “mucus”. The family is subdivided into four genera: Influenzavirus, Isavirus, Thogotovirus, and Quaranjavirus. The first genera contain viruses that cause disease in vertebrates, including birds, humans, and other mammals, while isaviruses are fish-infecting viruses (infectious salmon anemia virus).5 Thogotoviruses have been primarily associated with either hard or soft ticks and they have a wide geographic distribution. Moreover, they can infect several mammals but only a few cases have been reported of human infections and a novel thogotovirus (Bourbon virus) has only recently been associated with a febrile illness and death of a human patient in the United States in 2014.6 Quaranjaviruses predominantly infect arthropods and birds.7 Here we focus our attention on the influenza viruses, including influenza A, B, and C, which are of greater medical importance. The most common and also the most medically important of the influenza viruses are those designated type A, which infect humans and a wide array of other mammals, and predominantly birds. In particular, aquatic birds represent the wildlife reservoirs of the virus and play a role of paramount importance in the creation of human epidemic and pandemic influenza strains. Influenza B is not known to give rise to pandemics, and has a more limited host spectrum, in fact it has only recently been found to cause infections in seals, apart from man.8 Type C influenza viruses infect humans and swine, but only cause mild respiratory illness or no symptoms at all. More importantly, type C influenza viruses are not able to produce epidemics and, therefore, are of limited medical interest.
3. Virion structure and genomic architecture
Influenza viruses are pleomorphic, spherical particles (about 100 nm in diameter), although filamentous forms can occur. The virions are relatively unstable in the environment and influenza viruses are inactivated by heat, dryness, extremes of pH, and detergents. Virions are enveloped and their lipid membrane is derived from the host cell. The envelope lodges different glycosylated proteins that project from the surface of the virus. These proteins are the hemagglutinin (HA), the neuraminidase (NA), and the M2 ion channel proteins. The morphology of influenza A virus particles is, therefore, characterized by distinctive spikes, with lengths from 10 to 14 nm, which are readily observable in electron micrographs of virus particles. The approximate ratio between HA and NA is 4:1.
The matrix protein (M1) is situated just beneath the envelope, and underlying the M1 layer a helical superstructure, representing the core of the virus particle which is made of the ribonucleoprotein (RNP) complex, is observed. The RNP complex consists of the viral RNA segments, which are coated with the nucleoprotein (NP) and associated with the heterotrimeric polymerase complex (PB1, PB2, and PA) (Fig. 1.1). The chemical composition of virus particles is approximately 1% RNA, 5–8% carbohydrate, 20% lipid, and approximately 70% protein.9
The genomes of influenza A and B consist of eight separate negative-sense, single-stranded RNA segments known as viral RNAs. The influenza C genome has only seven segments. Each viral RNA segment exists as a structure known as viral ribonucleoprotein (vRNP) complex in which the RNA strand is wrapped by the NP and forms a helical hairpin that is bound on one end by a single heterotrimeric polymerase complex. Within the RNPs, RNA molecules account for the genetic material of the virus, NP monomers cover and protect it, whereas the polymerase complex is responsible for the transcription and replication.10 Noncoding sequences are present at both 5′ and 3′ ends of the viral gene segments; partially complementary sequences are located at the extreme termini which are highly conserved between all segments in all influenza viruses. When base-paired, these ends function as the viral promoter that is required for replication and transcription.
Each of the eight segments of the influenza A virus genome encodes for the viral proteins (Table 1.1). The whole influenza genome encodes for 10 major proteins, although alternative protein products have been characterized from several genome segments.11 The complete coding capacity of the influenza virus genome is only partially known and processes like splicing, and the use of alternative initiation codons or overlapping frames are employed by the virus to augment the variety of viral products that are generated during the infectious cycle. In fact, among the RNAs present, 2 of them (segment 7 and 8) are transcribed to messenger RNAs (mRNAs) which are then spliced into two chains encoding a further gene product each. Additionally, segment 2 presents at the 5′ end an alternative open reading frame that encodes for the 87 aa long PB1-F2 polypeptide. The full expression capacity of the influenza genome is further complicated by the abundant synthesis of small viral noncoding RNAs, whose roles in productive infections are still largely unknown.12,13
Schematic Representation of the Protein Products of Influenza A Virus Gene Segments
The three largest RNA segments each encode for one of the viral polymerase subunits, PB2, PB1, and PA.14 The influenza virus RNA-dependent RNA polymerase is a 250-kDa complex of these three genetic products. Electron microscopy studies have shown a compact structure with the three subunits tightly associated.15 PB1, representing the core of the complex and the most conserved of the polymerase subunits, contains the enzymatic motifs needed for RNA polymerization activity.16 As previously remembered, the second segment also encodes for an accessory protein, PB1-F2, from an alternate open reading frame within the PB1 gene. PB1-F2, which is unique to influenza A, has been found to localize to mitochondria and exhibiting a pro-apoptotic activity. The PB2 subunit recognizes and binds to the host mRNA cap structures generated by the cellular transcription machinery17,18 and the acidic protein PA has an endonucleolytic activity necessary for the viral cap-snatching process.19,20
Segment 4 codes for the HA protein. The mature HA protein is a trimeric type I integral membrane glycoprotein (with a single transmembrane span, N-terminus in the ectodomain and C-terminus in the cytosol) which is found in the lipid envelope of virions and on the surface of infected cells.21 HA is the most abundant proteic component of the viral envelope and is the major target for neutralizing antibodies. HA undergoes several posttranslational modifications including glycosylation,22,23 palmitoylation,24 proteolytic cleavage, disulphide bond formation, and conformational changes. Host cell proteases operate the maturation of the precursor HAO molecule into its HA1 and HA2 subunits (which are linked by a disulphide bridge) (Fig. 1.2, Part 1: HA1 in blue and HA2 in red) and this cleavage mechanism is essential for the fusion activity of HA. Moreover, HA is in charge of the binding of virions to host cell surface receptors (sialic acid).25
RNA segment 5 of influenza encodes RNA-binding protein NP.26 This is a highly basic protein whose main function is encapsidation of the viral RNA (an NP monomer of 56 kD binds 24 bases of RNA)27 leaving accessibility to the polymerase as a template for transcription.28
NP also plays a crucial role in transporting the viral RNPs into the nucleus.29,30 In fact, RNPs are too large to passively diffuse through the 9 Å nuclear pores.31 In order to shuttle RNPs in and out of nuclei and from the cytoplasm to the cell periphery, NP makes multiple viral and cellular protein associations involving distinct NP recognition sequences (NLSs, nuclear localization signals, and NES, nuclear export signals). During the late stages of infection, NP also associates with the cytoskeleton32 and NP has been found to bind to filamentous-actin.33
Segment 6 of influenza A encodes the NA protein which is the second major viral glycoprotein. This type II integral membrane protein (single transmembrane span, C-terminus in the ectodomain and N-terminus in the cytosol) is a tetramer and possesses critical sialidase (NA) activity that is needed for a productive release of viral particles from the infected cell.34
Segment 7 of influenza A encodes two proteins, the M1 and M2 protein. M1 is expressed from a collinear transcript, while M2 is derived from an alternative spliced mRNA. M1 associates with lipid membranes and plays an essential role in viral budding. It also regulates the movement of RNPs out of the nucleus and inhibits viral RNA synthesis at later stages of viral replication. M2 is a tetrameric type III membrane protein that has ion channel activity. It functions primarily during virus entry where it is responsible for acidifying the core of the particle which triggers dissociation of M1 from the viral RNPs (uncoating).
The shortest RNA (segment 8 in influenza A) encodes the NS1 protein from a collinear transcript and the NEP/NS2 protein from an alternatively spliced transcript. NS1 is a RNA-binding protein that is expressed at high levels in infected cells and is useful to inhibit host antiviral response, besides interfering with the host mRNA processing. The NEP/NS2 protein mediates the nuclear export of newly synthesized RNPs, corresponding with its expression at later times during viral infections.
Recently, several additions have been made to the list of gene products coded by the influenza A genome.35 As just described earlier in the paragraph, the PB1-F2 protein was discovered in 2001 and is encoded by an alternative open translation initiation sites near the 5′ end of the PB1 gene.36 Moreover, a third protein is made by the same mechanism by the PB1 gene, named PB1-N40. PB1-N40 is an N-terminal 39 aa polypeptide which is translated from the fifth AUG codon in frame with the PB1 start.37 In addition, segment 1, coding for PB2, produces a newly discovered viral protein, termed PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513–1894 of the PB2 mRNA is deleted.38 Novel polypeptides are also encoded by genomic segment 3 and are named PA-X, PA-N155, and PA-N182. The first of them is derived from a second open reading frame (X-ORF), accessed via ribosomal frameshifting39 and modulates host response.40 The other two (PA-N155 and PA-N182) do not have a polymerase activity, and have been found to be important for virulence and pathogenesis.41 Expression of specific spliced viral products throughout infection is also applied for two of the smallest segments, M1 and NS1. It was already known for a while that protein products from segment 7 included the matrix (M1) and ion channel (M2) proteins, made from a spliced transcript, but a further protein product with an alternative ectodomain and again by a splicing mechanism, has been recently identified, the M42.42 Finally, segment 8, besides the nonstructural (NS) protein NS1, also encodes a nuclear export protein NS2/NEP, and NS3 by alternative mRNA splicing.43
4. Viral replication
Influenza viruses bind to neuraminic acids (sialic acids) on the surface of cells to initiate the entry process25 (Fig. 1.3). Notwithstanding the ubiquitous nature of sialic acids, HAs of influenza viruses infecting different animal species show some preference for particular glycosidic linkages of the receptor. Human viruses preferentially bind to sialic acids linked to galactose by alfa 2,6 glycosidic bonds (SAα2,6Gal). On the other hand, avian viruses show a preference for alfa 2,3 glycosidic bonds (SAα2,3Gal).44,45 The preference of different influenza subtypes for diverse host-species is explained by the fact that SAα2,6Gal is found mostly in human trachea, while SAα2,3Gal is abundant in the gut epithelium of several bird species, but it should be noted that this represents a preference for receptor-antireceptor linkage and not an absolute specificity. In fact, avian and human cells contain sialic acids with both linkages, though differently expressed and distributed. In addition, high viral inoculum or adaptive point mutations in the HA gene may circumvent this problem.46 HA is able to perform both action of binding and mediation of fusion of juxtaposing membranes. HA is expressed as a trimeric rod-shaped molecule on the virion surface and is produced as a HA0 precursor which is then cleaved into two subunits HA1 (the globular more external part of the molecule) and HA2 (holding a transmembrane domain) by host cell proteases. The cleavage is needed for the full activity of the molecule. The major structural features of the HA trimer are: a long stem of triple-stranded coiled-coil of α-helices and a globular ectodomain exposed to the environment and derived from the HA1 portion of the monomers.47 The stalk region of HA connects the molecule to the virion envelope by a short hydrophobic sequence (transmembrane domain),25 while the globular head is responsible for the receptor binding through a receptor binding pocket located on its distal tip. The interaction between sialic acid and HA is considered to be of low affinity; therefore, in order to increase the total intensity of the interaction, several HA molecules cooperate to link more sialic acids molecules leading to a high-avidity binding on the cell surface.48 After binding to the receptor (Fig. 1.2, part 1), influenza viruses are taken up into the cell. Early imaging studies revealed that the virus enters the cell by receptor-mediated endocytosis.49–51 The main mechanism is still recognized to be by clathrin-mediated endocytosis, although a nonclathrin, noncaveolae pathway has been also attributed to influenza entry.52,53 Regardless of whether the influenza viruses are taken up by endocytosis or macropinocytosis, they end up exploiting the transport system via distinct endosomal stages and consequently changes in pH to release their viral ribonucleoprotein complexes (vRNPs) into the cytoplasm. After internalization by either uptake pathways, the virus initially localizes to early endosomes and then reaches late endosomes. A crucial function is devoted to proton pumps, which mobilitate protons from cytoplasm into the endosomal lumen; therefore, allowing for a gradual acidification of the endosome content.54,55 Initial lowering of pH in endosomes radically changes HA structure, which is rendered vulnerable to be digested by proteases. Consequently, HA is cleaved into the two subunits, connected together by disulphide bonds. Once the influenza virus is in the acidic environment of the late endosome, HA undergoes further conformational changes that expose the fusion peptide at the N-terminus of the HA2 and locate it toward the endosomal membrane.56,57 This in turn leads to the interaction of HA with the membranes of the endosome. Following the stepwise conformational changes, the fusion peptide is finally inserted into the target endosomal membrane (Fig. 1.2, part 2) and juxtaposition of the viral and endosomal membranes is reached.58–60 The crystal structures of pre- and postfusion HA (Fig. 1.2, part 1 and 3, respectively) have been solved, while only models based on such structures are available to predict the intermediate stages of the HA gradual conformational modifications. Furthermore, it has been demonstrated that several HA trimers (at least nine) are needed to execute membrane fusion by concurrent conformational changes to release the required energy.61 To proceed with fusion between viral and endosomal membranes with the increasing acidification, HA trimers tilt (Fig. 1.2, part 3) at the fusion site and a hemifusion stage is created where the outer leaflets of the interacting membranes are blended together and only the internal leaflets of the two membranes preserve the content from the virus and the endosome to mix together.62,63 Finally, both membranes fuse and a so-called fusion pore is established.64–66 As a consequence of these chain of processes, the influenza virus genetic material, in the form of a RNP complex, can be released through the fusion pore into the cytoplasm.67 Once fusion of viral and endosomal membranes has been achieved, uncoating of the viral capsid is necessary to release the VRNPs into the cytosol. This process appears to require the coordinated action of two viral proteins, namely M1 and M2. Considering influenza virion particle, M1 can be visualized by electron microscopy forming a structured stratum beneath the viral envelope.68,69 M1 serves to link the viral membrane containing the glycoproteins with the RNPs in the virus core through interactions between the middle domain of M1 with the NP on the RNP.70 During the uncoating process, the interactions of M1 with either the viral membrane or the vRNPs need to be released for allowing complete uncoating and subsequent transport of the RNPs into the nucleus. This requires the activity of the viral protein M2 which mediates the influx of H+ ions from the endosome into viral particle. This ion channel activity of M2 is regulated by pH, in fact, lowering the pH, the ion channel activity increases.71 Upon acidification of the endosome, M2 facilitates the proton influx from the endosome into the virion, leading to a pH values drop inside the virus particle.72 This change in pH inside the virus particle causes conformational changes in M1, therefore, the interaction between the RNPs and M1 is greatly weakened or lost pushing the release of the vRNPs into the cytoplasm.73,74 Finally the eight segments are transported as one moiety to the nucleus75 where RNA synthesis of influenza virus occurs. RNP complexes are not able to passively diffuse through nuclear pores (since the small size of the pores); therefore, proteins belonging to the family of α-importins recognize nuclear localization signals (NLS) on the NP protein and play an important role in the transport of RNA complexes.76 In order for the genome to be transcribed, it first must be converted into a positive sense RNA to serve as a template for the production of viral RNAs. Once in the nucleus, genomic ssRNA of negative polarity serves as a template for the synthesis of two different ssRNAs of positive polarity: (1) messenger RNAs (mRNAs), and (2) full-length complementary copies (cRNAs). Messenger RNAs are the incomplete copies of the template and they are capped and polyadenylated. The viral RNA-dependent RNA polymerase (RdRp) is made up of the three viral proteins: PB1, PB2, and PA. During transcription, viral RdRp interacts with the host polymerase II.77 PB2 shows endonuclease activity and binds to the 5′ methylated caps of cellular mRNAs cleaving the cellular mRNAs’ 10–15 nucleotides 3′ to the cap structure (this phenomenon is called “cap-snatching”). This cellular capped RNA fragment is then used by the viral RdRp to prime viral transcription.78–81 It is recognized that the three subunits of the polymerase act cooperatively and the synthesis of RNAs is a concerted action of all subunits. The synthesis of full-length complimentary copy (cRNA) of the vRNA does not need capped primer and the cRNA chain is not prematurely terminated and polyadenylated, as is the case of viral mRNA synthesis. The next step of replication of influenza genomic segments is the copying of vRNA on the template of positive strand cRNAs. This process also generates full-length products which assemble with NP and polymerase subunits and form RNP complexes. Such complexes are finally exported into the cytoplasm; the M1 protein and NS2 protein play a vital role in the translocation of these macromolecular structures.30 Packaging of eight different RNA segments in the form of RNPs into virion shells is a poorly understood phenomenon, though many models have been proposed.82–84 Some facts suggest the presence of packaging signals at both ends of the genomic segments; however, the precise sequences or structures responsible for specific packaging are still not well defined. All eight segments probably form a supercomplex before arrival at the plasma membrane binding sites.75 Before being directed into lipid rafts, these two glycoproteins are posttranslationally modified. The modifications take place in the endoplasmic reticulum and in the Golgi apparatus. In the endoplasmic reticulum, these proteins become correctly folded and glycosylated. They are also assembled into oligomers: HA into trimers and NA into tetramers. Capsids get assembled underneath the cell membrane where the inserted viral glycoprotein interacts with the matrix and the RNPs and the envelope virions exit by membrane budding. NA is required to remove sialic acids in order to allow the virus to leave its host cells.