Clinical outcomes for all serotypes can be unapparent or result in a spectrum of diseases ranging from self-limited dengue fever to severe dengue, a potentially lethal hemorrhagic illness. The incidence of dengue disease is growing as the mosquito vector spreads owing to urbanization, population growth, international travel, insufficient mosquito control efforts, and global warming. Although vaccines and antivirals are still unavailable to control DENV infections, a great effort is being made in this direction, and solutions will likely be accessible in the near future.
The structure of the DENV particle was solved through a combination of cryo—electron microscopy, imaging reconstruction, and X-ray crystallography 6 — 8. The particle comprises an electron-dense core surrounded by a lipid bilayer, in which two transmembrane viral proteins are inserted to form a glycoprotein shell. The core contains the nucleocapsid NC , formed by one copy of the single-stranded capped RNA genome in complex with multiple copies of the capsid protein.
DENV enters host cells by receptor-mediated endocytosis, which involves attachment and receptor binding by the viral E protein. Different mammalian and mosquito host proteins have been shown to interact with E at the cell surface, but a bona fide receptor for virus entry has not yet been identified for review, see 9.
The virus enters primarily through clathrin-mediated endocytosis 10 — Upon internalization and acidification of the endosome, fusion of viral and vesicular membranes mediated by conformational changes in the E protein allows NC release into the cytoplasm 16 , 17 ; for review, see 18 , Genome uncoating involves dissociation of capsid, which frees the RNA that is directly used for viral translation.
Viral protein synthesis takes place in the rough endoplasmic reticulum RER and renders a large polyprotein with a complex topology on endoplasmic reticulum ER membranes 20 — Most of the NS proteins are multifunctional. They provide enzymatic activities and render the proper environment for viral RNA replication, including remodeling cellular membranes and suppressing host antiviral responses.
Infection induces hypertrophy of intracellular membranes, which provides structures in which the genome is amplified 23 — RNA synthesis is catalyzed by the viral polymerase NS5 in a process that requires specific promoter recognition and genome cyclization 28 , The newly synthesized genome associates with capsid to form the NC, and this ribonucleoprotein complex buds into the ER lumen, acquiring the lipid bilayer, together with the viral E and prM proteins.
The immature viral particles travel through the secretory pathway. Furin-mediated proteolysis of prM in the trans Golgi network triggers rearrangement, homodimerization of E, and formation of mature viral particles, which are subsequently secreted Mature DENV capsid is a highly basic protein of 12 kDa that forms homodimers in solution with affinity for both nucleic acids and lipid membranes 31 , The residue monomer contains 26 basic amino acids and only 3 acidic residues.
The tridimensional structures of the DENV and WNV capsid proteins were solved by nuclear magnetic resonance and crystallography, respectively 33 , The dimer shows asymmetric charge distribution, with basic residues accumulating on one face of the molecule and a concave apolar surface on the opposite side.
The N-terminal domain is unstructured in solution and has a high density of positive charges 8 lysines or arginines in the first 22 residues.
Purification of recombinant DENV and YFV capsid proteins was associated with truncations of the N-terminal region, supporting the idea that this segment is flexible or structurally disordered. Accordingly, pioneering studies by Markoff and colleagues 37 described a conserved internal hydrophobic region spanning residues 45 to 65 of DENV4.
These authors showed that the mature capsid protein remains associated to ER membranes via this hydrophobic region, which is conserved in a wide range of mosquito- and tick-borne flaviviruses. Structure of the dengue virus DENV capsid protein. The hydrophobic cleft is indicated. Functional analysis has provided evidence that basic residues at the N-terminal region of DENV capsid also contribute to RNA binding and viral particle formation A model of RNA and lipid membrane binding of capsid is presented in Figure 1 b.
Capsid—RNA interaction studies have been hampered by the fact that capsid aggregates upon nucleic acid binding. In particular, positive charge neutralization by RNA interaction might drive aggregation through the hydrophobic region; however, experimental analyses of the aggregation process are still needed.
The RNA chaperone activity of WNV capsid was mapped to the C-terminal RNA-binding region of the protein 43 and was proposed to facilitate long-range interactions in the viral genome A hallmark of active RNA chaperone domains is a high content of basic residues that are structurally flexible, a property shared among all flavivirus capsid proteins; thus, capsid proteins from other members of the genus are expected to display the same RNA remodeling capacity.
Capsid is the first protein encoded in the viral genome, followed by prM Figure 2. These two proteins are connected by an internal hydrophobic signal peptide, known as anchor, which spans the ER membrane and is responsible for the translocation of prM into the ER lumen.
A model of a coordinated two-step proteolytic processing, at the N-and C-termini of anchor in the capsid—prM junction, has been proposed for different flaviviruses 47 — Matured capsid is released by proteolytic processing of the capsid—anchor junction by the viral NS3 protease, which requires the viral NS2B cofactor NS2B In the ER lumen, the host signal peptidase cleaves the anchor—prM junction.
Membrane topology of dengue virus structural proteins. The orientation of the structural proteins across the ER membrane is shown. Transmembrane helices are indicated by cylinders, and the sites of posttranslational cleavage by signal peptidase are indicated by red arrows.
The cleavage site of the viral NS2B-3 protease is indicated by a green arrow and the furin cleavage site by a blue arrow. It has been demonstrated that the anchor peptide is not efficiently recognized by the signal peptidase in the ER and that, for certain flaviviruses, cleavage at the cytoplasmic side by the NS2B-3 protease allows peptide accommodation for efficient cleavage of anchor—prM.
Mutagenesis within anchor that increased peptidase processing uncoupled the sequential order of the two cleavages but impaired viral particle formation Thus, the accepted model suggests that capsid protein maturation triggers prM maturation and particle assembly in a timely and spatially coordinated process.
NCs contain a single molecule of the viral genome and multiple copies of capsid but lack a defined symmetry 6 , 7 , Thus, the specific manner in which capsid directs NC formation remains unclear.
Capsid—RNA binding has been proposed to be nonspecific and mainly driven by electrostatic interactions. Viral RNA synthesis occurs in replication complexes that contain membranous structures formed by ER invaginations, known as vesicle packets VPs. It has been shown that these vesicles have necks open to the cytoplasm, through which the newly synthesized RNA exits Interestingly, replication complexes have been observed by using transmission electron microscopy and electron tomography of DENV-infected cells as physically linked to capsid-containing ER membranes.
A model has been proposed wherein the viral genome is transported directly to sites of NC assembly at the ER membranes, and the budding particles in the ER lumen acquire the lipid bilayer, E, and prM 23 , 24 , 26 Figure 3. NC incorporation into the budding particle is not driven by interactions between the NC and the cytoplasmic domains of E or prM inserted into the viral membrane. Structural studies using DENV particles described a low-density gap between the density contributed by the NC and that contributed by the lipid bilayer without evidence of a contact between the NC and E or M 6 , 7.
Budding of viral particles into the ER is NC independent, because empty flavivirus particles lacking capsid and viral RNA can be produced by overexpressing only prM and E proteins In this regard, it has been shown that viral RNAs are not encapsidated if they were not actively synthesized in replication complexes Model of dengue virus assembly. The budding viral particle, containing the nucleocapsid, the viral proteins E and prM, and lipid membranes, is shown within the ER.
Abbreviations: C, capsid protein; E, envelope protein; ER, endoplasmic reticulum; NS, nonstructural protein; prM, pre—membrane protein. The sequential order of cleavages at the C—prM junction possibly enhances NC uptake into budding membranes In this respect, the presence of the NS2B-3 active protease required for capsid maturation may play additional roles in coordinating genome recruitment Figure 3.
NS3 also contains RNA helicase and RNA annealing activities and interacts with the viral genome; thus, this protein may be the missing link between the viral genome and capsid for NC formation. A genetic link between NS2A and NS3 for viral particle formation has been described, suggesting that particle assembly uses a complex system that includes different host and viral components Moreover, a recent report showed that viruses with specific mutations within NS1, which were still competent for replication, release up to fold fewer infectious DENV particles than the parental virus, providing novel evidence for a function of NS1 in viral particle assembly Further studies are necessary to understand the complex network of proteins involved in genome recruitment and to elaborate more comprehensive models of viral NC assembly.
The coding sequence for capsid contains a number of RNA structures necessary for viral genome replication, limiting the genetic manipulation of protein residues. Incorporation of mutations within capsid must take into account potential effects on viral RNA synthesis. A systematic analysis including structure prediction and biochemical probing of the complete capsid-coding RNA was recently reported Recently reported strategies include duplication of specific RNA structures or duplication of the complete capsid-coding sequence 38 , Although capsid is the least conserved of the flavivirus proteins, the structural properties are very similar and the charge distribution is well conserved.
A multiple-sequence alignment of mosquito-borne flavivirus capsid proteins indicates low sequence conservation. Dots indicate conserved residues, and dashes indicate deletions. The red text indicates basic residues in the capsid protein of DENV2. Pink boxes highlight conserved residues along the different mosquito-borne flaviviruses. The figure shows the alignment of these consensus sequences, performed using Geneious 3.
Mutational studies were performed using reporter DENV systems to uncouple cis -acting RNA structures from the capsid-coding sequence 38 , In contrast, deletion of the basic-rich N-terminal sequence of capsid impaired DENV particle formation.
Interestingly, differential requirements were noticed for infections in mosquito and human cells A systematic mutational analysis indicated that at least two positive charges in each of the two clusters were necessary for viral particle assembly in human cells.
On the basis of these studies, an accumulation of positive charges rather than of residues in specific positions was proposed to be crucial for DENV particle formation. Mutations in other regions of DENV capsid were also reported. However, pseudo-revertants with extended deletions of capsid from amino acid 40 to 76 were recovered in culture. These results indicated that a large deletion of approximately 36 amino acids was better tolerated than a small deletion of 4—7 amino acids in the hydrophobic region, suggesting that a short version of capsid could form NCs by an alternative mechanism.
Remarkable flexibility in capsid protein function has been observed among various flaviviruses. In tick-borne encephalitis virus TBEV , infectious viruses were still recovered even after deletion of up to 16 residues in the central region of capsid between positions 28 and 48 Also, TBEV with deletions ranging from 19 to 30 residues long in a hydrophobic region resulted in viruses with second-site mutations that increased the hydrophobicity of the protein Interestingly, viral mutants lacking 16 amino acids of capsid were found to be attenuated but very immunogenic in adult mice Studies using a YFV replicon trans -packaging system demonstrated that large deletions in the N- and C-terminal regions of capsid were also tolerated for particle formation This observation provides evidence that one of the two proposed RNA-binding regions is sufficient for NC assembly of this virus, in contrast with observations on DENV In conclusion, capsid proteins from different flaviviruses tolerate extensive deletions and mutations, suggesting that they do not require a defined 3D structure for their function but rather rely on basic residues to recruit the viral RNA.
Nevertheless, distinct flavivirus capsid proteins show different degrees of tolerance for structural changes; DENV capsid is one of the least tolerant proteins in this regard. Although DENV particle assembly occurs in the cytoplasm, capsid has been detected in both the cytoplasm and the nucleus of infected cells 70 — 75 Summary Figure. Inside the nucleus, capsid accumulates in nucleoli, whereas in the cytoplasm it is distributed between ER membranes and the surface of lipid droplets LDs This distribution has been observed early after DENV infection, suggesting that it is not a consequence of cell damage during viral replication Dengue virus particle assembly, capsid protein interactions, and subcellular distribution in an infected cell.
The newly synthesized viral genome exits the VPs and is recruited by the capsid protein green to form the NC, which buds into the ER and acquires lipid membranes and the structural viral proteins E and prM.
The capsid protein is also distributed in different cellular compartments, ER membranes, nucleoli, and lipid droplets. However, the functional significance of the fraction of capsid associated with the nucleus and LDs is still unclear.
Capsid subcellular distribution could be temporally and spatially controlled during DENV infection. In this regard, mutations in capsid that lead to protein mislocalization during infection greatly inhibit viral RNA synthesis This observation is puzzling because, although deletion of the complete capsid-coding sequence does not affect viral RNA synthesis, point mutations that alter its localization do.
Removal of mature capsid from ER sites, near RNA replication, could be important to avoid premature capsid interaction with the viral RNA; thus, sequestration of the protein in the nucleus or LDs could be a mechanism to regulate protein availability during the viral life cycle.
If proteolytic maturation of capsid—anchor is associated with NC assembly and particle budding, it is possible that ER-associated capsid is used for NC formation and capsid localized in nucleoli or LDs plays auxiliary functions during infection.
We next summarize the available data on DENV capsid subcellular localization. Transport of capsid into the nucleus has been proposed to be mediated by nuclear localization signals NLSs. Using protein overexpression, it was originally proposed that capsid nuclear localization was predominantly due to the bipartite sequence 72 ; however, studies using DENV2-infected cells with mutations in the three putative NLSs of capsid showed a reduction of nuclear localization for mutants in each of the three sites This study suggested a lack of correlation between capsid nuclear accumulation and viral propagation in cell culture.
Thus, the functional significance of the nuclear-associated capsid during viral infection remains unclear. Nuclear localization of capsid from other flaviviruses has also been observed. Using JEV as a model, capsid accumulation in the nucleus was dependent on amino acids G42 and P43, both in mammalian and infected insect cells. Mutations of these residues resulted in a reduction of JEV pathogenesis in mice and lower titers in cell culture These residues are conserved among different flaviviruses Figure 4 , but whether they are also involved in the nuclear accumulation of capsid from other members of the genus remains to be seen.
The capsid residues involved in translocation were the consensus sequence of a bipartite NLS located between residues 85 and and amino acids 42 and It is important to bear in mind that the predicted NLSs are patches of basic residues in the capsid protein that are also involved in RNA binding.
Therefore, further analysis of mutant viruses is necessary to dissociate DENV capsid requirements for nuclear localization and NC assembly. In particular, single substitutions of residues L50 or L54 in the hydrophobic cleft were sufficient to abrogate capsid accumulation on LDs and to reduce viral particle formation. In vitro studies using atomic force microscopy provided evidence that a peptide corresponding to the disordered N-terminal region of capsid interacts with negatively charged LDs, suggesting that this region also facilitates LD binding Further studies indicated that the N-terminal peptide inhibits, in a dose-dependent manner, in vitro binding of capsid to LDs It has also been proposed that capsid binding to LDs depends on high concentrations of potassium and that this binding could be mediated by the LD-associated protein TIP47 Studies to define the significance of capsid on LDs during viral infection are complicated by the fact that hydrophobic residues involved in LD accumulation may also be important for ER membrane association.
Thus, the lack of viral particle formation for mutant viruses with substitutions in the hydrophilic region may be due to defects in viral morphogenesis. But in these, the total number of subunits is always a multiple of In some animal viruses, the nucleocapsid is surrounded by a membrane, also called an envelope.
This envelope is made up of a lipid bilayer and is made up of lipids from host cells. It also contains virus-encoded proteins, often glycoproteins that are transmembrane proteins. These viral proteins serve many purposes, such as binding to receptors in the host cell, playing a role in membrane fusion and cell entry, etc.
They can also form channels on the viral membrane. Many enveloped viruses also contain matrix proteins, which are internal proteins that bind the nucleocapsid to the envelope. They are very abundant that is, many copies per virion and are generally not glycosylated.
Some virions also contain other non-structural proteins that are used in the viral life cycle. Examples of this are replicases, transcription factors, etc. These nonstructural proteins are present in low amounts in the virion. Enveloped viruses are formed by budding through cell membranes, usually the plasma membrane, but sometimes an internal membrane such as ER, Golgi, or nucleus. In these cases, the assembly of viral components genome, capsid, matrix occurs on the inner side of the membrane, the envelope glycoproteins clump together in that region of the membrane, and the virus breaks out.
This ability to bud allows the virus to leave the host cell without lysing or killing the host. Conversely, unenveloped viruses and some enveloped viruses kill the host cell to escape. Your email address will not be published. Save my name, email, and website in this browser for the next time I comment. Helical viruses can be enveloped or naked. The first virus described, tobacco mosaic virus, is a naked helical virus. In fact, most plant viruses are helical, and it is very uncommon that a helical plant virus is enveloped.
In contrast, all helical animal viruses are enveloped. These include well-known viruses such as influenza virus, measles virus, mumps virus, rabies virus, and Ebola virus Fig. A Vesicular stomatitis virus forms bullet-shaped helical nucleocapsids. Fred A. B Tobacco mosaic virus forms long helical tubes. C The helical Ebola virus forms long threads that can extend over nm in length. Of the two major capsid structures, the icosahedron is by far more prevalent than the helical architecture.
In comparison to a helical virus where the capsid proteins wind around the nucleic acid, the genomes of icosahedral viruses are packaged completely within an icosahedral capsid that acts as a protein shell. Initially these viruses were thought to be spherical, but advances in electron microscopy and X-ray crystallography revealed these were actually icosahedral in structure.
An icosahedron is a geometric shape with 20 sides or faces , each composed of an equilateral triangle. An icosahedron has what is referred to as 2—3—5 symmetry , which is used to describe the possible ways that an icosahedron can rotate around an axis. If you hold an icosahedral die in your hand, you will notice there are different ways of rotating it Fig.
A helix is mathematically defined by two parameters, the amplitude and the pitch, that are also applied to helical capsid structures. The amplitude is simply the diameter of the helix and tells us the width of the capsid. The pitch is the height or distance of one complete turn of the helix. In the same way that we can determine the height of a one-story staircase by adding up the height of the stairs, we can figure out the pitch of the helix by determining the rise , or distance gained by each capsid subunit.
A staircase with 20 stairs that are each 6 inches tall results in a staircase of 10 feet in height; a virus with This is the architecture of tobacco mosaic virus. Your pencil would be right in the middle of a triangle facing up and a triangle facing down. If you rotate the icosahedron clockwise, you will find that in degrees you encounter the same arrangement symmetry : a triangle facing up and a triangle facing down.
Continuing to rotate the icosahedron brings you back to where you began. This is known as the twofold axis of symmetry, because as you rotate the shape along this axis your pencil , you encounter your starting structure twice in one revolution: once when you begin, and again when rotated degrees. On the other hand, if you put your pencil axis directly through the center one of the small triangle faces of the icosahedron, you will encounter the initial view two additional times as you rotate the shape, for a total of three times.
This is the threefold axis. Similarly, if your pencil axis goes through a vertex or tip of the icosahedron, you will find symmetry five times in one rotation, forming the fivefold axis. It is for this reason that an icosahedron is known to have 2—3—5 symmetry, because it has twofold, threefold, and fivefold axes of symmetry. This terminology is useful when dealing with an icosahedral virus because it can be used to indicate specific locations on the virus or where the virion has interactions with the cell surface.
For instance, if a virus interacts with a cell surface receptor at the threefold axis, then you know this interaction occurs at one of the faces of the icosahedron. A protein protruding from the capsid at the fivefold axis will be found at one of the vertices tips of the icosahedron. All of the illustrations of viruses in Fig.
How many twofold axes of symmetry are found in one icosahedron? How about the number of threefold or fivefold axes? How many faces, edges, and vertices are found in an icosahedron?
A Icosahedron faces fuchsia triangles , edges red rectangles , and vertices violet pentagons are indicated on the white icosahedron. B The twofold axis of symmetry occurs when the axis is placed through the center of an edge. The threefold axis occurs when the axis is placed in the center of a face C , and the fivefold axis passes through a vertex of the icosahedron D.
Viral proteins form each face small triangle of the icosahedral capsid. Viral proteins are not triangular, however, and so one protein subunit alone is not sufficient to form the entire face.
Therefore, a face is formed from at least three viral protein subunits fitted together Fig. These can all be the same protein, or they can be three different proteins. The subunits together form what is called the structural unit.
The structural unit repeats to form the capsid of the virion. A Virus capsids are composed of viral protein subunits that form structural units.
The triangulation number T indicates the number of structural units per face of the icosahedron. The red lines outline a triangular face of the icosahedron, while the purple pentagons indicate the vertices fivefold axes of the icosahedron. But how can some viruses form very large icosahedral capsids? The answer is repetition. The structural unit can be repeated over and over again to form a larger icosahedron side. The number of structural units that creates each side is called the triangulation number T , because the structural units form the triangle face of the icosahedron.
The geometry and math involved with icosahedral capsid structure can be complex, and only the very basics are described here. In any case, by increasing the number of identical structural units on each face, the icosahedron can become progressively larger without requiring additional novel proteins to be produced. Some viruses have triangulation numbers over 25, even! The proteins that compose the structural unit may form three dimensional structures known as capsomeres that are visible in an electron micrograph.
In icosahedral viruses, capsomeres generally take the form of pentons containing five units or hexons containing six units that form a visible pattern on the surface of the icosahedron See Fig. Capsomeres are morphological units that arise from the interaction of the proteins within the repeated structural units. Why does the icosahedral virus structure appear so often? Research has shown that proteins forming icosahedral symmetry require lesser amounts of energy, compared to other structures, and so this structure is evolutionarily favored.
Many viruses that infect animals are icosahedral, including human papillomavirus, rhinovirus, hepatitis B virus, and herpesviruses Fig. Like their helical counterparts, icosahedral viruses can be naked or enveloped, as well. Poliovirus A , rotavirus B , varicella—zoster virus C , the virus that causes chickenpox and shingles, and reovirus D.
Note that C is enveloped. The majority of viruses can be categorized as having helical or icosahedral structure. A few viruses, however, have a complex architecture that does not strictly conform to a simple helical or icosahedral shape. Poxviruses, geminiviruses, and many bacteriophages are examples of viruses with complex structure Fig.
Poxviruses, including the viruses that cause smallpox or cowpox, are large oval or brick-shaped particles — nm long.
The geminiviruses also exhibit complex structure. As their name suggests, these plant-infecting viruses are composed of two icosahedral heads joined together. Bacteriophages , also known as bacterial viruses or prokaryotic viruses , are viruses that infect and replicate within bacteria.
Many bacteriophages also have complex structure, such as bacteriophage P2, which has an icosahedral head, containing the nucleic acid, attached to a cylindrical tail sheath that facilitates binding of the bacteriophage to the bacterial cell. Vaccinia virus A , a virus belonging to the poxvirus family, has a complex capsid architecture with a dumbbell-shaped core.
Geminiviruses B have a double-icosahedron capsid. Bacteriophages, such as P2 C , often have complex capsid structure. The classification of viruses is useful for many reasons. It allows scientists to contrast viruses and to reveal information on newly discovered viruses by comparing them to similar viruses. It also allows scientists to study the origin of viruses and how they have evolved over time. The classification of viruses is not simple, however—there are currently over different viral species with very different properties!
One classification scheme was developed in the s by Nobel laureate David Baltimore. The Baltimore classification system categorizes viruses based on the type of nucleic acid genome and replication strategy of the virus.
As will be further discussed in the next chapter, positive-strand also positive-sense or plus-strand RNA is able to be immediately translated into proteins; as such, messenger RNA mRNA in the cell is positive strand.
Negative-strand also negative-sense or minus-strand RNA is not translatable into proteins; it first has to be transcribed into positive-strand RNA. Baltimore also took into account viruses that are able to reverse transcribe , or create DNA from an RNA template, which is something that cells are not capable of doing.
Together, the seven classes are. There are a variety of ways by which viruses could be classified, however, including virion size, capsid structure, type of nucleic acid, physical properties, host species, or disease caused. Because of this formidable challenge, the International Committee on Taxonomy of Viruses ICTV was formed and has been the sole body charged with classifying viruses since Taxonomy is the science of categorizing and assigning names nomenclature to organisms based on similar characteristics, and the ICTV utilizes the same taxonomical hierarchy that is used to classify living things.
It is important to note that viruses, since they are not alive, belong to a completely separate system that does not fall under the tree of life. Whereas a living organism is classified using domain, kingdom, phylum, class, order, family, genus, and species taxa singular: taxon , or categories, viruses are only classified using order, family, genus, and species Table 2.
The ICTV classifies viruses based upon a variety of different characteristics with the intention of categorizing the most similar viruses with each other. The chemical and physical properties of the virus are considered, such as the type of nucleic acid or number of different proteins encoded by the virus.
0コメント