These processes result in viruses able to evade the long-term adaptive immune responses in many hosts. The influenza A, B, and C viruses, representing three of the five genera of the family Orthomyxoviridae , are characterized by segmented, negative-strand RNA genomes. Sequencing has confirmed that these viruses share a common genetic ancestry; however, they have genetically diverged, such that reassortment — the exchange of viral RNA segments between viruses — has been reported to occur within each genus, or type , but not across types.
Influenza A viruses are further characterized by the subtype of their surface glycoproteins, the hemagglutinin HA and the neuraminidase NA. Influenza viruses have a standard nomenclature that includes virus type; species from which it was isolated if non-human ; location at which it was isolated; isolate number; isolate year; and, for influenza A viruses only, HA and NA subtype.
While many genetically distinct subtypes — 16 for HA and 9 for NA — have been found in circulating influenza A viruses, only three HA H1, H2, and H3 and two NA N1 and N2 subtypes have caused human epidemics, as defined by sustained, widespread, person-to-person transmission [ 1 ].
By electron microscopy, influenza A and B viruses are virtually indistinguishable. They are spherical or filamentous in shape, with the spherical forms on the order of nm in diameter and the filamentous forms often in excess of nm in length.
The influenza A virion is studded with glycoprotein spikes of HA and NA, in a ratio of approximately four to one, projecting from a host cell—derived lipid membrane [ 1 ]. A smaller number of matrix M2 ion channels traverse the lipid envelope, with an M2:HA ratio on the order of one M2 channel per 10 1 2 HA molecules [ 2 ].
The envelope and its three integral membrane proteins HA, NA, and M2 overlay a matrix of M1 protein, which encloses the virion core. However, influenza C virions are compositionally similar, with a glycoprotein-studded lipid envelope overlying a protein matrix and the RNP complex. The influenza C viruses have only one major surface glycoprotein, the hemagglutinin-esterase-fusion HEF protein, which corresponds functionally to the HA and NA of influenza A and B viruses, and one minor envelope protein, CM2 [ 1 ].
See Table 1. The eight segments of influenza A and B viruses and the seven segments of influenza C virus are numbered in order of decreasing length.
Segment 7 of both influenza A and B viruses code for the M1 matrix protein. The genomic organization of influenza C viruses is generally similar to that of influenza A and B viruses; however, the HEF protein of influenza C replaces the HA and NA proteins, and thus the influenza C virus genome has one fewer segment than that of influenza A or B viruses.
Kindly provided by Megan L. The ends of each vRNA segment form a helical hairpin, which is bound by the heterotrimeric RNA polymerase complex; the remainder of the segment is coated with arginine-rich NP, the net positive charge of which binds the negatively charged phosphate backbone of the vRNA [ 13 — 15 ].
However, the extreme ends of all segments are highly conserved among all influenza virus segments; these partially complementary termini base-pair to function as the promoter for vRNA replication and transcription by the viral polymerase complex. The noncoding regions also include the mRNA polyadenylation signal and part of the packaging signals for virus assembly. A segmented genome enables antigenic shift , in which an influenza A virus strain acquires the HA segment, and possibly the NA segment as well, from an influenza virus of a different subtype.
This segment reassortment can happen in cells infected with different human and animal viruses, and the resulting virus may encode completely novel antigenic proteins to which the human population has no preexisting immunity.
Characterization of the reconstructed influenza virus indicated that its unique constellation of genes was responsible for its extreme virulence, with the HA, NA, and PB1 genes all contributing to its high pathogenicity [ 16 , 17 ]. Influenza viruses recognize N-acetylneuraminic sialic acid on the host cell surface. Sialic acids are nine-carbon acidic monosaccharides commonly found at the termini of many glycoconjugates.
Thus, they are ubiquitous on many cell types and in many animal species. The differential expression of sialic acids in the mammalian respiratory tract may help to explain the low infectivity but high pathogenicity of some avian strains. The lungs are not as accessible to airborne virus particles as is the upper respiratory tract nasopharynx, paranasal sinuses, trachea, and bronchi , so avian virus infection is relatively rare in humans.
The crystal structure of the HA molecule is a trimer with two structurally distinct regions: a stem, comprising a triple-stranded coiled-coil of alpha-helices, and a globular head of antiparallel beta-sheet, positioned atop the stem [ 25 ]. The head contains the sialic acid receptor binding site, which is surrounded by the predicted variable antigenic determinants, designated A, B, C, and D in the H3 subtype [ 26 ] and Sa, Sb, Ca1, Ca2, and Cb in the H1 subtype see Figure 1 [ 1 ].
During virus replication, the HA protein is cleaved by serine proteases into HA1 and HA2; this post-translational modification is necessary for virus infectivity. The HA2 portion is thought to mediate the fusion of virus envelope with cell membranes, while the HA1 portion contains the receptor binding and antigenic sites reviewed in [ 27 ]. Antibodies to HA neutralize virus infectivity, so virus strains evolve frequent amino acid changes at the antigenic sites; however, the stem-head configuration of the HA molecule remains conserved among strains and subtypes.
These relatively minor changes accumulate in a process called antigenic drift. Eventually, mutations in multiple antigenic sites result in a virus strain that is no longer effectively neutralized by host antibodies to the parental virus, and the host becomes susceptible again to productive infection by the drifted strain. The head contains the sialic acid receptor-binding site, which is surrounded by the five predicted antigenic sites Sa, Sb, Ca1, Ca2, and Cb.
The stem comprises helices A and B and the fusion peptide, as shown. Adapted from a figure, kindly provided by James Stevens and Ian Wilson, in [ 1 ]. Following attachment of the influenza virus HA protein or the HEF protein of influenza C virus to sialic acid, the virus is endocytosed.
The acidity of the endosomal compartment is crucial to influenza virus uncoating in two ways. Second, hydrogen ions from the endosome are pumped into the virus particle via the M2 ion channel. The M2 protein, a transmembrane ion channel found only in influenza A virus, has portions external to the viral envelope, along with the HA and NA.
The M2 protein is the target of the amantadine class of anti-influenza drugs, which block ion channel activity and prevent virus uncoating [ 30 , 31 ]; also, because it is a surface protein, it has been proposed as a vaccine component [ 32 , 33 ].
Internal acidification of the influenza virion via the M2 channel disrupts internal protein-protein interactions, allowing viral RNPs to be released from the viral matrix into the cellular cytoplasm [ 34 ]. The nucleus is the location of all influenza virus RNA synthesis — both of the capped, polyadenylated messenger RNA mRNA that acts as the template for host-cell translation of viral proteins, and of the vRNA segments that form the genomes of progeny virus.
Unlike host cell mRNA, which is polyadenylated by a specific poly A polymerase, the poly A tail of influenza virus mRNA is encoded in negative-sense vRNA as a stretch of five to seven uracil residues, which the viral polymerase transcribes into the positive sense as a string of adenosines that form the poly A tail [ 36 — 38 ].
The envelope proteins HA, NA, and M2 are synthesized, from mRNA of viral origin, on membrane-bound ribosomes into the endoplasmic reticulum, where they are folded and trafficked to the Golgi apparatus for post-translational modification. All three proteins have apical sorting signals that subsequently direct them to the cell membrane for virion assembly. Although comparatively little is known about the translation and sorting of the non-envelope proteins, M1 is thought to play a role in bringing the RNP-NEP complex into contact with the envelope-bound HA, NA, and M2 proteins for packaging at the host cell membrane [ 1 ].
Influenza virus is not fully infectious unless its virions contain a full genome of eight segments or seven segments, for influenza C virus. Learn More. Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination.
These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney MDCK cells. The cell cycle stage of MDCK cells had no impact during early infection.
Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination HA titer.
The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps.
The online version of this article doi Influenza is a contagious respiratory disease caused by influenza virus infections. Annually, influenza A and B viruses account for three to five million cases of severe illness and between , and , people decease due to these infections worldwide estimates of the World Health Organization WHO , Fact sheet Influenza No. Despite numerous attempts to develop drugs for the treatment of severe influenza infections, only two classes of antiviral drugs have been licensed so far.
However, due to their frequent use, resistant influenza virus variants have already emerged. Thus, the best protection against influenza is annual vaccination. Nowadays, cell culture-based influenza vaccine production has become an important alternative to the conventional manufacturing process in embryonated chicken eggs.
Compared to egg-based processes, cell culture technology has significant advantages such as higher flexibility and scalability since it is independent from the timely supply and laborious handling of embryonated eggs. In addition, these processes are not vulnerable to the threat of avian influenza viruses that can kill laying flocks.
Moreover, sterility can easier be maintained in cell culture reviewed by Audsley and Tannock Recently, the first recombinant influenza vaccine produced in insect cells received approval Buckland et al. However, a thorough understanding of the viral replication cycle, virus-host cell interactions, and virus spreading in cell populations is crucial to further optimize vaccine production in these cells.
Influenza viruses belong to the family of Orthomyxoviridae. They are enveloped viruses with a segmented single-stranded RNA genome of negative polarity.
The genome of influenza A viruses consists of eight segments encoding for 10 major proteins and additional seven accessory polypeptides reviewed by Vasin et al. The RdRP accomplishes both transcription and replication of the viral genome, which takes place in the nucleus of infected cells. While transcription is directly initiated after the nuclear import of vRNPs, replication can only proceed in the presence of newly synthesized viral proteins. At later stages of the infection cycle, the matrix protein 1 M1 and the nuclear export protein NEP; also known as non-structural protein 2 NS2 bind to vRNPs which leads to their nuclear export and to the termination of viral RNA synthesis reviewed by Cros and Palese Subsequently, viral genomes and proteins are transported to the plasma membrane where virus assembly and budding take place reviewed by Bouvier and Palese Activation of signal transduction pathways and accumulation of viral components in the course of an infection lead to the induction of programmed cell death apoptosis.
On the one hand, virus propagation can be impaired when apoptosis is induced early during infection before virus replication has reached its full magnitude. On the other hand, apoptosis at later time points seems to support the release of virions and therefore becomes pro-viral reviewed by Herold et al. Many details of the influenza virus life cycle have already been unraveled, but still little is known about the relative importance of all steps involved and how their interplay determines the virus titer in cell cultures.
To identify possible bottlenecks for the production of influenza viruses on the molecular level, we thoroughly studied the dynamics of influenza virus replication in adherent MDCK cells.
To this end, we analyzed virus release kinetics in a single-cycle infection by exchanging the medium in regular intervals. Using electron microscopy, we compared the morphology of virus particles from an early phase of the infection with particles from the later stage when almost exclusively noninfectious particles were produced.
In addition, we examined the intracellular dynamics of viral RNA synthesis and the localization of viral components in the course of an infection cycle. The latter was done by imaging cytometry that combines the throughput and sensitivity of conventional flow cytometry with the spatial resolution of fluorescence microscopy. Finally, we performed an infection at low multiplicity of infection MOI to investigate if cells in a particular cell cycle stage become preferentially infected and support enhanced influenza virus replication.
The seed virus titer was determined by standard plaque assay 1. Thereafter, the inoculum was removed, cells were washed twice with PBS, and 13 mL of infection media were added to the flasks.
At every sampling time point, one flask was used to analyze the intracellular infection dynamics by quantitative reverse transcription PCR RT-qPCR and imaging flow cytometry.
To study virus release kinetics, only 5 mL of virus infection media were added to one flask. The supernatant of this flask was harvested every 4 h. To this end, the flask was rocked to swirl up detached cells and the supernatant was harvested.
Five milliliters of infection media was used to wash the cell monolayer and was then combined with the supernatant. Fresh infection media 5 mL was added to the flask that was returned to the incubator. We then determined the cell number in the supernatant i.
Four flasks containing 13 mL of infection media served as controls to determine the virus titer without medium exchange and to obtain the cell count of adherent cells. In addition, low MOI infections were performed to investigate if cells in a certain cell cycle stage become preferentially infected. One day before infection, 2. At each sampling time point, one T25 flask was harvested. Virus titers were determined by the hemagglutination assay Kalbfuss et al.
Cell-specific, cumulative virus release was assessed by referring to the maximum cell count obtained in each experiment. Infected cells in T75 flasks were rocked to swirl up detached cells and the supernatant was harvested.
Detached cells were separated from the supernatant by centrifugation. Remaining adherent cells in T75 flasks were trypsinized and afterwards combined with the detached cells from the previous step. The T7 promotor sequence was introduced by primers Online Resource, Tab. Primer sequences are listed in supplementary table S2 Online Resource. To calculate viral RNA concentration, C t values obtained for the RNA reference standards were plotted against log 10 numbers of viral molecules resulting in a linear calibration curve.
Following incubation, the cells were washed three times with FACS buffer. Secondary antibody staining was performed using Alexa Fluor conjugated polyclonal goat anti-mouse antibody LifeTechnologies, A at a dilution of For nuclear staining, 0. For infection experiments at low MOI, up to , single cells were measured.
One thousand cells of these samples were acquired for compensation with the respective compensation settings. IDEAS software version 6. Compensation matrices were generated using the corresponding compensation files. Only single cells in-focus were selected for analysis. Segmentation masks for M1- and vRNP-positive cells were generated based on mock-infected samples.
CH1- and Ch5-double positive cells were plotted on histograms using this feature. The M1 analysis was performed using the same procedure, but taking the corresponding channels into account. Apoptotic cells were quantified based on chromatin condensation and nuclear fragmentation leading to an increase in the intensity and a decrease in the area of the nuclear signal as well as cell shrinkage leading to higher BF contrast as described before Maguire et al.
Infected cells were identified using a Max Pixel histogram of mock-infected samples for each time point. An imaging service using negative stain transmission electron microscopy nsTEM was performed by Vironova Stockholm, Sweden.
After blotting off the stain, the grid was allowed to air dry. For each sample, five remotely located grid positions, with good sample embedding, were selected. At each position, by traversing the grid at 68 k magnification, a total of 20 images were acquired.
Image data was collected, as soon as one or more particles were detected in the field of view. Particles were subsequently classified as intact, deformed, or broken, and large membranous by manual image analysis.
Thereby, spherical and close to spherical particles approximately 80— nm in diameter with well-resolved surface spike proteins were classified as intact particles. Particles that displayed major perturbations and even rupture of the membrane were classified as deformed or broken particles. Moreover, particles that had the characteristic appearance of lipid membranes folded structures, commonly with bright edges and a denser interior and were larger than the typical influenza virion in size — nm were classified as large membranous particles.
This high MOI ensures that the complete cell population becomes rapidly infected and consequently enables the detailed analysis of a synchronized single-cycle infection. We investigated the dynamics of virus production by harvesting the complete supernatant of the cells and subsequent addition of fresh medium every 4 h.
All samples were subjected to the TCID 50 assay and the HA assay to determine the concentration of infectious and total virus particles, respectively. Cumulative growth curves were generated by adding up the titers of every successive sampling interval in the course of the infection experiment Fig.
To check whether the replacement of the medium every 4 h had an impact on the infection process, cells were infected under the same conditions but incubated without medium exchange for 12, 24 and 36 h. No significant differences in the total virus particle counts were observed between cells with and without medium exchange demonstrating that the experimental procedure did not affect the influenza virus propagation.
In contrast, infectious virus particles reached lower final yields of about virions per cell Fig. When we calculated the cell-specific virus particle production per hour, it became obvious that the rate of virus release for both infectious and total virus particles reached its maximum already in the sampling interval between 4 and 8 h post infection hpi Fig.
In the following 12 h, the virus release occurred at a rather constant rate and the ratio of infectious to total virus particles was more or less stable. At later stages of the infection, cells almost exclusively produced noninfectious particles below 0.
In addition, we determined cell counts and the cell viability in the time course of the single-cycle infection. Simultaneous to the decrease in the production of infectious particles, the adherent MDCK cells started to detach from the bottom of the cell culture flask and the viability measured by trypan blue staining started to decrease Fig. Most of the cells were found in the supernatant at 28 hpi when the production of total virus particles also declined.
Recent improvements in imaging and RNA labeling techniques have made it possible to monitor the entire entry process in single cells 61 , 62 , 83 — A striking observation from these studies is the efficiency with which the eight vRNAs reach the nucleus, indicating how effectively vRNPs recruit the host nuclear import factors. While the bulk of the vRNP trafficking work has been carried out using various immortalized cell lines, the potential species related differences, and the essential role of vRNP trafficking in reassortment, emphasize the need for further methodology development to examine the details of IAV entry in primary cells and tissue explants.
The nucleotide complementation locks the vRNA template into the polymerase active site within the PB1 subunit and results in the formation of an A—G dinucleotide from which the cRNA is elongated Upon exiting the polymerase, the cRNA associates with newly synthesized NP molecules and a single copy of the viral polymerase to assemble into a cRNP The transient nature of the rNTP annealing and dinucleotide formation makes it technically challenging to exclude either possibility.
The PB2 cap-binding domain then rotates to position the newly acquired capped primer into the PB1 catalytic center where it is extended using the vRNA as a template This process likely involves multiple cycles of dissociation, repositioning, and reannealing of the mRNA to this template region of the vRNA to achieve polyadenylation. Cap binding positions the region of the mRNA 10—13 nucleotides downstream for cleavage by the endonuclease domain in the PA subunit.
Following the priming event, the viral polymerase extends the mRNA transcript. The initial mRNAs are transcribed by the vRNP-associated polymerases and exported from the nucleus for translation by cytoplasmic ribosomes However, the M and NS transcripts also possess donor and acceptor splice sites that match well with those in human transcripts These sites recruit the cell spliceosome, which produces the spliced transcripts that encode for the M2 and NS2 proteins, respectively — The NS transcript has been reported to maintain a similar ratio of non-spliced and spliced transcripts throughout infection , whereas the ratio of the spliced M transcripts encoding M2 have been shown to increase during infection These observations imply that NS1 and NS2 are always equally expressed, while M2 expression is more biased toward the later stages of infection.
IAV protein synthesis is entirely dependent on the translation machinery of the host cell. Following nuclear export [reviewed in Ref. Coordination of viral ribonucleoprotein vRNP assembly and trafficking to the plasma membrane. The vRNP trafficking either occurs by Rabcontaining vesicles associated with microtubules or xii b through Rab11 located in the modified endoplasmic reticulum ER membranes.
How the vRNPs reach the budding site at the plasma membrane is currently not known. The viral RNA-binding protein NS1 is synthesized early and also imported into the nucleus, where it can act as an inhibitor of interferon signaling [reviewed in Ref. NS2 alternatively known as the nuclear export protein and M1 are imported into the nucleus as well. Multiple studies have implicated these two proteins in the nuclear export of vRNPs 70 , 71 , — Within the cytoplasm the vRNPs are trafficked toward the plasma membrane for viral assembly by Rab Rab11 facilitates the interaction by associating with the viral polymerase PB2 subunit , potentially providing a quality control mechanism that ensures new virions incorporate vRNPs carrying a polymerase.
While several studies have indicated that specific vRNP associations likely contribute to the packaging of the eight vRNPs 35 , , , the underlying mechanisms remain to be established. The IAV membrane proteins, which are ultimately destined for the viral envelope, are synthesized by ribosomes associated with the ER membrane. Linked to the dependence on SRP, mutations that alter the targeting sequence hydrophobicity of cellular secretory proteins have been shown to decrease their ER targeting and subsequent synthesis , Although this aspect has not been examined for the IAV membrane proteins, there is evidence that the hydrophobicity of their ER-targeting sequences change , , which suggests IAVs potentially use this mechanism to titrate NA and HA expression.
NA contributions to viral release and intercellular movement. Following synthesis, the proteins oligomerize and are trafficked through the Golgi to the plasma membrane. Box A The envelope protein NA promotes release of the virus from the infected cell surface by hydrolyzing the glycosidic bond attaching the SAs. Box C NA can cleave off the SAs from the glycoproteins within the mucus to facilitate movement of the virus to neighboring cells.
The translocon enables passage of the elongating NA, HA, and M2 polypeptides into the ER lumen and facilitates the membrane partitioning of their respective TMD segments through a lateral gate , To activate the membrane integration, the TMD segments have to be of the appropriate length and hydrophobicity , The uncharacteristic hydrophobicity loss was shown to be possible because of the NA TMD being positioned at the N-terminus One function of the glycans is to increase the folding efficiency of NA and HA by recruiting the lectin chaperones calnexin and calreticulin and the associated oxidoreductase ERp57, which aids in disulfide bond formation , — This is especially crucial for the HA and NA proteins that possess a significant number of intramolecular disulfide bonds e.
By contrast, M2 possesses two intermolecular disulfide bonds in its tetrameric conformation Oligomerization of HA involves the trimerization of independently folded monomers, whereas NA tetramerization has been proposed to result from the pairing of two co-translationally formed dimers, which assemble through a process involving the N-terminal TMD of NA , In line with this model, it has been shown that the TMD is essential for proper NA folding, and that the decreasing hydrophobicity in the N1 TMDs functions to support the folding and oligomerization of the enzymatic head domain , IAVs easily achieve the protein concentration-dependent requirement for oligomerization due to the abundance of HA and NA that is synthesized during an infection.
However, these high synthesis levels at the ER can also be deleterious by activating the ER-stress response. Despite everything that is known about the synthesis and assembly of the IAV membrane proteins, several aspects have yet to be addressed. These include obtaining atomic structures of full-length HA and NA in a membrane, something that should become easier to address with the advances in cryo-electron microscopy structure determination. Identifying if the NA protein removes SA residues directly from substrates within the Golgi , as this could decrease the effectivity of nonmembrane permeable NA inhibitors.
It is also unclear how IAVs regulate the timing and expression levels of the viral proteins as viral mRNA transcription shows little temporal variation , While it is likely that M2 is regulated in part by splicing , , this does not apply to HA and NA.
However, a clear mechanistic picture for influenza protein regulation is lacking. The cleavage occurs in either a monobasic, or a multibasic, cleavage site Multibasic sites are commonly found in highly pathogenic avian IAVs and are cleaved by furin, a calcium-dependent serine endoprotease that is located within the trans-Golgi network Furin is also ubiquitously expressed , which is one of the major reasons why avian IAVs with a multibasic cleavage site are generally more pathogenic.
By contrast, human and low pathogenic avian IAVs encode for HAs with a monobasic cleavage site, which have been shown to be processed by different proteases in human respiratory epithelial cells.
HAT localizes at the plasma membrane where it can either cleave newly synthesized HA or the HA found in cell-associated virions , The M2 ion channel is thought to prevent the premature activation of HA following cleavage by equilibrating the slightly acidic pH of the Golgi , Distinct from furin, TMPRSS2 expression has been found to be more restricted to the upper and lower respiratory tract, whereas HAT was mainly shown to be expressed in the upper respiratory tract These cell tropisms suggest that lower respiratory infections are likely mediated by TMPRSS2, and could be one of the primary reasons human IAVs are confined to the epithelial layer of the respiratory tract.
HA is believed to localize to these distinct regions based on fatty acid modifications of the C-terminal cysteine that occur in the Golgi — , whereas NA enrichment has previously been attributed to a property in the C-terminus of the TMD In contrast, M2 has been shown to accumulate at the boundaries of these budding domains , and the cytosolic protein M1 has been proposed to localize to the budding region by associating with the short cytoplasmic tails of HA and NA However, it is equally plausible that NA and HA create membrane domains with a unique lipid profile that have a high affinity for M1.
Finally, the vRNPs, delivered to the cell periphery by Rab11, are thought to localize to the budding site by binding to M1 , In addition to orchestrating the assembly of the correct viral components at the apical budding site, IAVs also have to remodel the membrane to induce bud formation, and ultimately scission of the viral envelope from the plasma membrane.
To promote bud formation, the virus must first induce significant curvature in the membrane and then constrict the two opposing membranes of the viral envelope to help to facilitate membrane scission. Based on cumulative data regarding budding, IAVs appear to induce membrane curvature through a combination of these mechanisms. Indicative of using molecular crowding and bending proteins, several studies have demonstrated that HA and NA expression is sufficient to induce budding, and that the efficiency and shape uniformity benefit from the presence of M1 — These results indicate that the abundance of HA and NA on one side of the membrane can contribute to curvature.
It also is intriguing to speculate that the asymmetric shape of NA plays a role in this process as it is often seen clustering in the viral membrane 16 , By contrast, M1 appears to be analogous to a membrane-bending protein as it recruited to the cytosolic side of the membrane budding site, oligomerizes upon reaching the membrane, and these oligomers have been modeled to form curved structures — Based on these properties, it is plausible that M1 significantly influences the membrane curvature at the budding site, potentially explaining its role in discerning whether IAVs form spheres or filaments 27 , The ion channel M2 localizes to the budding site boundary and has also been shown to contribute to IAV scission by functioning as a membrane-bending protein , With this domain positioned in the cytosol, the intercalation results in negative membrane curvature, which has been proposed to facilitate viral bud neck formation and scission, presumably by decreasing the distance between the two opposing membranes of the viral envelope While much of the framework concerning IAV budding has been established, it has been difficult to identify the details of the budding process, in part due to the mobility and heterogeneity of the plasma membrane.
The lack of strong phenotypes from domains proposed to contribute to budding could also imply that IAVs have built redundancy into the budding process — The possibility of redundancy is certainly plausible, as IAVs contain the necessary components to allow for a combination of lipid recruitment, molecular crowding, and a membrane-bending protein.
Once the newly assembled IAVs bud, their release is highly dependent on the sialidase activity of NA. NA is a homotetramer, and each subunit is comprised of a short N-terminal cytoplasmic tail six amino acids , followed by a TMD, a length variable stalk, and a globular enzymatic head domain The catalytic Tyr residue is found in a highly conserved active site that forms a deep pocket in the center of each monomer All of the residues necessary for catalysis exist within each monomer , which has made it difficult to reconcile why NA evolved to function as a tetramer , , Structures of the enzymatic head domain indicate that NA tetramers bind up to five calcium ions and calcium has been shown to contribute to NA activity , , However, it remains unclear why influenza NA has evolved to position a calcium ion at the tetrameric interface.
NA facilitates viral release by catalyzing the hydrolysis of the glycosidic linkage that attaches SA to underlying sugar molecules — However, a thorough analysis of NA SA preference is lacking. More recent studies have found that some strains possess NAs that are inefficient enzymes, but still capable of SA binding, raising the question of whether a poor NA enzyme could contribute to, or replace, the HA receptor-binding function , The movement of IAVs from cell to cell in the respiratory epithelium is significantly different from that in immortalized cell lines grown in liquid culture due to the presence of different cell types and a mucus layer.
The mucus layer provides a protective barrier for the epithelium and is rich in heavily glycosylated mucins that can interact with IAVs and limit cell binding , Recent work showed that this function may also apply to transmission, as IAVs that possess low NA activity, and are inhibited by mucus, are deficient in aerosol and contact transmission IAVs are constantly exposed to negative and positive selection pressure, which shapes how the virus evolves.
The functional requirements of each IAV protein, such as enzyme catalysis, substrate binding, oligomerization, and domains that perform essential interactions with host proteins all combine to create substantial negative selection pressure that often manifests in the form of sequence conservation.
Negative pressure can also come from functions within the vRNA sequences. In addition, the exposure of IAVs to the immune response and constantly changing environments such as host, temperature, pH, cell type, and antivirals result in positive selection pressure.
Experimentally, addressing each type of selection has its caveats, but clearly a holistic picture of both IAV and host functions are required to begin predictions of evolutionary constraints on the virus. Most studies on the influenza evolutionary process focus primarily on antigenic drift and antigenic shift. However, all the viral transcribed RNAs are subject to replication errors by the viral polymerase, which are estimated at 1 per 2,—10, nucleotides — Consequently, both the viruses and the viral proteins are likely to exist as large heterogeneous populations during an infection.
As many IAV proteins are homo-oligomers this can potentially generate heterogeneity within individual protein complexes that could have functional advantages.
By applying single particle and single cell analysis, these types of aspects are beginning to be investigated Another interesting approach is deep mutational scanning, which has been used to examine the site-specific amino acid tolerance of IAV proteins in general, and in the context of different selection pressure — Currently, the best characterized protein in IAVs is HA, which has two primary functions, i to initiate binding to the host cell and ii to deliver the vRNPs to the host cell cytosol by fusing the viral and endosomal membranes.
The receptor-binding site responsible for entry is located in the considerably larger HA1 subunit that is known to be immunodominant, explaining the high sequence variability in this region By contrast, the smaller HA2 subunit, containing the fusion peptide that is necessary to deliver the viral genome to the host cell, shows considerably higher sequence conservation.
This organization is logical from the viral perspective as the large HA1 subunit likely blocks antibody recognition of HA2. The viral downside is the need to escape antibodies that inhibit the receptor-binding pocket without losing specificity and the binding function. Based on this knowledge, several exciting new strategies are being developed to elicit the production of antibodies that target the more conserved region of HA — The hope is that these strategies will generate broadly neutralizing antibodies that recognize multiple HA subtypes from IAVs and the distinct lineages in IBVs, providing longer lasting immunity and alleviating the threat of potential pandemics.
A similar approach using NA would likely provide additional benefits. However, our knowledge of NA lags behind HA. Currently, it is still not known why NA has evolved to function as a tetramer, which is relevant because this property presumably restricts the potential antigenic drift mutations it can accommodate and still function.
It should also be considered that changes in RBPs have been associated with various cancers, which could possibly influence the susceptibility to influenza infections , With the growing interest in RNA biology, this aspect of IAV infections is likely to receive considerable attention in the future. In terms of IAV antivirals, the recent progress in determining the structures and mechanisms of the viral polymerase should significantly aid in the current development of drugs aimed at inhibiting different aspects of IAV transcription Through continued progress in defining the fundamental mechanisms that are necessary for IAV infections, replication and intercellular movement, it should become possible to minimize the annual burden caused by IAVs.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U. Front Immunol. Published online Jul Author information Article notes Copyright and License information Disclaimer. Reviewed by: Bernard A. Specialty section: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.
Received Apr 17; Accepted Jun The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC. Abstract Influenza viruses replicate within the nucleus of the host cell. Keywords: influenza A virus, viral ribonucleoprotein, hemagglutinin, viral entry mechanism, viral envelope proteins, HA and NA, viral replication, neuraminidase. Influenza Viruses Influenza viruses belong to the Orthomyxoviridae family and are classified as either type A, B, C, or the recently identified type D 1 , 2.
Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Perspectives IAVs are constantly exposed to negative and positive selection pressure, which shapes how the virus evolves. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Footnotes Funding. References 1. Characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the Orthomyxoviridae family.
MBio 5 :e31— Palese P, Shaw ML. Fields Virology. Palese P, Schulman JL. Mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes. Influenza virus genome consists of eight distinct RNA species. Houser K, Subbarao K. Influenza vaccines: challenges and solutions. Cell Host Microbe 17 — An overlapping protein-coding region in influenza A virus segment 3 modulates the host response.
Science — A complicated message: identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA. J Virol 83 — Identification of a novel viral protein expressed from the PB2 segment of influenza A virus. J Virol 90 — Influenza research database: an integrated bioinformatics resource for influenza virus research.
Nucleic Acids Res 45 :D— J Virol 87 — A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7 — Evolution and ecology of influenza A viruses. Microbiol Rev 56 — Curr Top Microbiol Immunol — Detection of evolutionarily distinct avian influenza A viruses in Antarctica. MBio 5 :e— Evidence for the introduction, reassortment, and persistence of diverse influenza A viruses in Antarctica.
Influenza virus pleiomorphy characterized by cryoelectron tomography. PLoS One 9 :e Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem —9. The structure of native influenza virion ribonucleoproteins. Science —7. Organization of the influenza virus replication machinery. Science —4. Structure of influenza A polymerase bound to the viral RNA promoter. Nature — Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter.
J Gen Virol 74 Pt 7 — Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Filamentous influenza viruses. Curr Clin Microbiol Rep 3 — Genetic studies of influenza viruses. Viral morphology and growth capacity as exchangeable genetic traits.
Rapid in ovo adaptation of early passage Asian strain isolates by combination with PR8. J Exp Med — Spherical influenza viruses have a fitness advantage in embryonated eggs, while filament-producing strains are selected in vivo.
Reverse genetics studies on the filamentous morphology of influenza A virus. J Gen Virol 84 — The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle formation. Virology — Structural organization of a filamentous influenza A virus. Conserved and host-specific features of influenza virion architecture. Nat Commun 5 Cellular proteins in influenza virus particles.
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