Influenza Virus : A brief review of mechanisms
Maintained by Amit ChattopadhyayAbstract
Influenza viruses are orthomyxoviruses and are characterized by mutability and high frequency of genetic reassortment. This leads to tremendous changes in the antigenic nature of the viral surface glycoproteins. Mainly, there are three types of Influenza viruses i.e. type A, B and C. Of these, type A virus has highest antigenic variability resulting in its antigenic shift and antigenic drift, properties that are major problems for defence and developing vaccine against this virus. Type C virus is antigenically most stable. These single stranded RNA viruses are surrounded by a lipid envelop with two virus encoded glycoproteins which play an important role in entry of the virus into the host cell through membrane fusion. Influenza virus, which has eight separate segments of negative stranded RNA in its genome, is also unusual among the RNA viruses with respect to replication as all of its RNA transcription and replication occurs within the nucleus of the infected cells. It continues to challenge the scientific world by devising a novel way to avoid the activity of PKR (Protein Kinase RNA activated) which is one of the interferon-induced cellular proteins synthesized for host cell defense against invasion.
The human Influenza A Virus
Introduction
Influenza is an acute respiratory illness caused by infection with influenza virus. Outbreaks of the infection are very common throughout the world and these result in significant morbidity in the general population and in increased mortality in high risk patients. The influenza virus infects vertebrates.
Typical features of the viruses are shown in table 1.
Table 1
Typical features of influenza viruses
|
Feature |
Characteristic |
|
Genome organization |
Single stranded RNA in eight pieces |
|
Inner Ribonucleoprotein helix |
9 nm in diameter |
|
RNA in nucleocapsid |
RNase sensitive |
|
Fusion of virus with cell |
Endosome |
|
Transcription of viral RNA |
Host cell nucleus |
|
Genetic reassortment |
Frequent |
|
Rate of antigenic change |
High |
Taxonomy
Influenza viruses belong to the Orthomyxoviridae family. Influenzavirus A and B contain human and animal strains of influenza type A and human strains of influenza type B. Influenzavirus C contains human strains of influenza type C virus and also Swine strains. Classification of the viruses into A, B, and C is allowed by the antigenic differences of the two internal structural proteins i.e. nucleocapsid (NP) and matrix proteins (M). These proteins have no cross-reactivity among the three types. Sub-typing of the virus is done by the antigenic variations in the surface glycoproteins, HA and NA.
The standard nomenclature system for influenza virus isolates, includes the following information: type, host of origin, geographic origin, strain number and year of isolation. Further to this, the antigenic descriptions of the glycoproteins are given in parentheses for type A. For human isolates, the host of origin is not indicated. An example is: a/Hong Kong/03/68(H3N2). If the name of the host is included, it will be at the start as in: A/swine/Iowa/15/30(H1N1).
Till date, about 14 subtypes of HA (i.e. H1 - H 14) and nine subtypes of NA (i.e. N1 - N9) have been recovered in different combinations, from birds, animals and humans. Three HA subtypes (H1 - 3) and two NA subtypes (N1, N2) have been recovered from humans.
The taxonomic structure of the family includes:
Structure of the Virus
Usually, the influenza virus is spherical and 100nm in diameter. However, they display a lot of variation in their size.
Morphology
The virus particles contain nine different structural proteins in the form of nucleoproteins (helical in configuration and 9nm in diameter). There are three large proteins (PB1, PB2 and PA) that are bound to the viral ribonucleoprotein. These are responsible for RNA transcription and replication of the virus. Further to this, there is the matrix protein (M1) which encloses the particle underneath the lipid envelope. The matrix protein contributes to 40% of the viral protein and is considered to be important in morphogenesis of the particle.
The virions are enveloped with a lipid envelop which is derived from the cell. Pleomorphic and filamentous forms are known to occur. They may be spherical, or filamentous; 50-120 nm in diameter, or 20 nm in diameter and about 200-300(-3000) nm long.
There are distinct surface glycoprotein {Neuraminidase (NA) and hemagglutinin (HA)} projections of envelope about 500 spikes projecting 10-14 nm from the surface. These are dispersed evenly over all the surface (i.e. haemagglutininesterase (HEF)), or dispersed equally over all the surface, but the various types are in clusters i.e. haemagglutinin (HA) the major glycoprotein is interposed irregularly by clusters of neuraminidase (NA). The ratio of HA to NA is about 4-5 : 1. Overall, HA represents about 25% of viral protein and NA represents about 5%. On the envelope, the M2 ion channel protein is also present. This protein, however, is present in low quantities, at only a few copies per particle.
Nucleocapsids of the influenza viruses are enclosed within lipoprotein membrane. The nucleoproteins are of different size classes with loop at each end. Information about the arrangement within the virion is uncertain at the moment. The Nucleocapsids are filamentous; with no clear modal length (of different size classes), and have dimensions of 50-130 nm in length and 9-15 nm in diameter. They have a helical symmetry.
The Genome
Virions contain 7 segments influenza C virus to 8 segments (influenza A and B virus) of linear negative-sense single stranded RNA. Total genome length is 12000-15000 nt. The largest segment among these is 2300-2500 nt long. The second largest is 2300-2500 nt; third 2200-2300 nt; fourth 1700-1800 nt; fifth 1500-1600 nt; sixth 1400-1500 nt; seventh 1000-1100 nt; and the eighth is 800-900 nt. The genome sequence has terminal repeated sequences which are repeated at both ends. Terminal repeats are at the 5'-end and are 12-13 nucleotides long. Nucleotide sequences of 3'-terminus are identical. The Nucleotide sequences are the same within the genera of same family and most are on RNA (segments), or on all RNA species. Terminal repeats at the 3'-end 9-11 nucleotides long. The encapsidated nucleic acid is solely genomic. Defective interfering copies may be present in each virion. The overall size of the genome is at 13.6 kb.
Most of the segments of the virus genome code for a single protein. For many influenza viruses, the whole genome is now known. The first 12 - 13 nucleotide sequences at each end of the genomic segment are important for transcription of the virus and are conserved among all eight RNA segments.
Genetic reassortment of the virus results from intermixing of the parental gene segments in the progeny of the viruses when a cell is co-infected by two different viruses of a given type. This phenomenon is facilitated by the segmental nature of the genome of influenza virus. Genetic reassortment is manifested as sudden changes in the viral surface antigens.
Structure - function interplay
Hemagglutinin (HA) of the virus
The HA protein binds virus particles to susceptible cells. It is also the major antigen against which neutralizing antibodies are directed by the host defense system. Thishas tremendous clinical significance as it is primarily due to variability of this protein, that allows for continuous evolution of new strains and causes influenza epidemics around the world.
Completed amino acid sequence for HA has been calculated from the sequence of cloned DNA copies of the HA gene and using X-ray crystallographic techniques, three-dimensional structure of the protein has been elucidated. The primary sequence of HA contains 566 aminoacids. A short signal sequence at the amino terminus inserts the polypeptide into the endoplasmic reticulum. The HA protein is cleaved into two sub-parts i.e. HA1 and HA2. These are associated tightly through a disulfide bridge. A hydrophobic stretch near the carboxyl terminal of HA2 anchors the HA molecule in the membrane with a short hydrophilic tail extending into the cytoplasm. Oligosaccharide residues are added at several sites. The whole HA molecule is foolded into a complex structure and each linked HA1 and HA2 dimer forms an elongated stalk capped by a large globule. The base of the stalk anchors it in the membrane. The HA spike on the virus particle has been shown to be a trimer, composed of three intertwined HA1 and HA2 dimers. The trimerization results in greater stabilization of the spike. The cllular receptor binding site is a pocket located at the top of each large globule.
A clevage separates HA1 and HA2 and is considered necessary for infectiousnes of theparticle. This may occur intracellularly or extracellularly by cellular proteases. Virus particles which are not thus cleaved can also attach to the cell receptors, but are not infectious.
Neuraminidase (NA) of the virus
The antigenicity of NA on the surface of the influenza virus is also important in determining the subtype of the influenza virus isolates. The complete sequence of NA is also known. Unlike HA, the spike on the virus particle is a tetramer, composed of four identical monomers. A thin stalk is topped with a head shaped like a box. There is a catalytic site for action of neuraminidase on the top of each head. Therefore, each spike of NA contains four active sites.
The function of NA is mainly at the end of the life cycle of the virus. A sialidase enzyme removes the sialic acid from glycoconjugates and facilitates the release of the virus particles from from the infected cell surfaces during the budding processes and this prevents self aggregation of virions by removing sialic acid residues from viral glycoproteins. It has also been suggestd that NA helps the virus to wade through the mucin layer in the respiratory tract to reach the epithelial cells, which are the target cells for the virus.
Antigenic Drift and Antigenic Shift
As has been mentioned earlier, frequent changes in HA and NA allow the influenza virus to have tremendous variability. This is important for the survival of the virus in hostile environments in the host. Since the host defense mounts an antibody response towards the antigens of the parent viral antigens, the new antigenic virus can survive easily due to lack of the hostile response. The epidemiology of influenza is essentially a story of the dog-fight between host defense and its dodging by the influenza virus through generations. This has also been the most difficult problem in making of a vaccine against influenza.
Antigenic drift is the term used to indicate minor antigenic variations in HA and NA of the influenza virus from the original parent virus, while major changes in HA and NA which make the new virions significantly different, are called Antigenic shift. The difference between the two phenomena is a matter of degree.
Antigenic drift (minor changes) occurs due to accumulation of point mutations in the gene which results in changes in the amino acids in the proteins. Sequence changes, occuring this way can alter the antigenic site on the molecule. It is understood that a variant must sustain two or more mutations before a significantly new (epidemiologically significant) variant strain emerges.
Changes which are extreme, and drastic (too drastic to be explained by mutation alone) result in antigenic shift of the virus. It has been seen that the segmented genomes of the influenza viruses reassort readily in double infected cells. Genetic reassortment between human and non-human influenza virus has been suggested as a mechanism for antigenic shift. This bears reference to the fact that Influenza B and C viruses have not been seen to exhibit antigenic shift (because few related viruses exist in animals).
Entry of the virus into cells
Influenza virus enter the host cells by a process of membrane fusion. This may occur at the cell plasma membrane or within the endocytic vacoular system. The exact mechanism is dependent upon the characteristics of the virus fusion proteins. Fusion of endocytosed viruse may be triggered by the acidic pH present within the endocytic pathway, which causes specific conformational changes in the spike protein. Upon binding to cell surface sialic acid residues on glycoproteins and glycolipids, the virus undergoes endocytosis via coated pits and vesicles and is then delivered to endosomes. HA is the protein which is central to both the receptor binding and fusion function of the virus. Binding of influenza virus to the cell surface is readily reversible due to the action of Viral NA. Released virions, may then rebind and the cycle may then go on. Within the endosome, acidic pH triggers virus-endosome membrane fusion.
Environmental survival of the virus
Influenza viruses are known to be resistant to the environment. They can be stored at 0-4o C form many weeks withgout any loss of their viability. The viruses, however, lose their infectivity rapidly at lower temperatures of -20o C (compared with at, say 4o C). The viruses are resistant to inactivation of their infectivity and hemagglutination at alkaline pH compared to acidic pH (more susceptible to acidic pH).
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