Togavirus model (Pov-Ray)
Togavirus model 2
togavirus section
RNA Viruses
Pre-requisites: read Virus_Tech page 1.

Many viruses carry RNA rather than DNA as their genetic material. This RNA may be single or double-stranded.
Like DNA viruses, RNA viruses come in a wide variety of forms. The model above is a togavirus (such as Semliki
Forest Virus (SFV) or Sindbis virus).The core of the virus is the nucleocapsid - single-stranded RNA (ssRNA)
packaged inside a protein capsid with icosahedral symmetry (not an icosahedron, in this instance as there are
more than 20 triangular faces, but the symmetry is icosahedral).
RNA packaged inside the togavirus capsid; note that
the capsid is fenestrated (it has 'windows').
The nucleocapsid is partially covered by a coat of C protein and enveloped by a phospholipid bilayer viral
membrane )derived from the host cell's membrane) which contains the viral protein spikes.

Adhesion and Injection of DNA

The protein spikes adhere to specific receptors on the target cell surface-membrane. The nucleocapsid, with
its C protein shell enters the host cytosol and the RNA is released and escapes from the capsid.

Replication

The replication of the RNA and the protein capsid and their packaging are summarised in the diagram below.
The single-stranded RNA is a plus or positive-strand, meaning that it is of the right sense to be immediately
transcribed directly into mRNA. [Some ssRNA viruses carry negative-strand RNA, which must first be copied
into the complementary (+)-strand which is then transcribed.] The (+)-RNA contains two START (AUG)
codons and transcription from each of these results in the synthesis of a different set of proteins.
Togavirus replication
In the early phase of infection, translation proceeds from the first AUG codon, resulting in the synthesis of a
long polyprotein (using the host's ribosomes) which is then cleaved into separate early protein polypeptides
which fold into enzymes and proteins needed  for RNA replication, producing the complementary (-)-strand
RNA, using the original (+)-strand as a template.

Part of this (-)-strand is then copied to produce a short (+)-strand mRNA which lacks the first START codon.
This is translated from the second START codon, producing a different polyprotein which is cleaved to
produce the
late proteins which form the virus particle, and include the C protein. In the meantime, the
(-)-RNA is copied repeatedly to produce lots of (+)-strand RNA genomes which are packaged into the
assembling virions. The nucleocapsid is assembled in the host cytosol, where the C protein coat is added.
Spike proteins are manufactured by the rough endoplasmic reticulum and Golgi apparatus of teh host cell
before being added to the host cell-surface membrane, where they form patches (excluding host proteins) to
which the assembled virions add and bud from the cell, acquiring their envelope as they do so.
Viroids

Viroids are plant pathogens that consist of circular ssRNA of about 300b (220 to 380 bases, the smallest is
220b). E.g. potato spindle tuber viroid (PSTVd) which causes infectious disease in potato plants. They code
for no proteins and have no protein capsid or protein-coat at all: they are naked infectious RNA molecules
and thus one of the simplest parasites conceivable. Their replication requires RNA polymerase II, provided
by the host cell and occurs by
rolling circle replication. The RNA has a 2D/3D structure, with some
regions pairing by hydrogen-bonding between the bases to give double-stranded regions, and the molecule
is rod-shaped. Some are
ribozymes, meaning that they fold to form structures with enzymatic activity, such
as catalysis of cutting the concatemers (produced by rolling circle replication) into individual viroids.
Viroid
Subviral Satellites

Hepatitis D virus (HDV) is the smallest known animal virus and has as mall genome of circular
single-stranded RNA or (-)sscRNA of 1.7 kb, in which about 70% of the nucleotides pair with other bases in
the molecule, forming a rod-like molecule. This virus is not able to complete its life-cycle without a
helper
virus
, in this case hepatitis B virus (HBV). HDV must coinfect the same cell as HBV in order to complete its
development as it requires some of the HBV genes. A virus like HDV is called a satellite virus (or subviral
satellite) and is thus parasitic on HBV. It is possible that HDV evolved from a viroid or similar infectious RNA
(it is thought that undiscovered viroids may infect and cause disease in animals).

Like a viroid, the HDV RNA has regions that can form catalytically active ribozymes, which are required to cut
the concatemers produced during genome replication (by (+)RNA to (-)RNA rolling-circle replication, which
first requires synthesis of the (+)RNA antigenome from the original (-)RNA genome) into individual genomes.
This is the fastest known self-cleaving ribozyme in nature, cutting the RNA in less than one second. Neither
encode their own RNA polymerase, required for RNA genome replication, but use host RNA polymerase.

The HDV genome encodes only two proteins: the large and small delta antigens (HDAg-S and HDAg-L) from
a single open reading frame (ORF). HDAg-S is produced early in infection and is required for viral
replication. HDAg-L appears in the later stages and inhibits viral replication and instead promotes viral
particle assembly.

Classification of Viruses

The Baltimore scheme classifies viruses into the following groups, based upon the nature of their genomic
material (different sources number the groups in different orders):

1.
dsDNA viruses, with linear dsDNA: e.g. adenovirus (icosahedral symmetry), T4 bacteriophage (binary
symmetry) and poxviruses (complex structure), or with circular dsDNA: e.g.hepatitis B virus.

2.
ssDNA, e.g. parvoviruses (icosahedral) such as adenoassociated virus.

3.
dsRNA, e.g. reovirus (icosahedral).

4.
(+)ssRNA, e.g. togaviruses (icosahedral), coronaviruses.

5.
(-)ssRNA, e.g. rhabdoviruses (helical).

6. RNA, with a DNA step in replication, e.g. retroviruses (e.g. HIV-1).
Influenza
Inluenza RNP
Influenza

Influenza virus is a (-)ssRNA virus whose genome is divided into 8 separate chromosomes (unusual for a
virus!). Influenza B, which infects humans, has a genome size of 14.648 kb and forms particles which are
spherical to filamentous). These 8 RNA segments are packaged into 8 helical ribonucleoprotein particles
(RNPs) enclosed in a protein capsid (made of the
matrix or M1 protein) surrounded by a phospholipid
bilayer
envelope (with the M protein lining the inside of the envelope). Embedded in the envelope are two
types of glycoprotein spike:
haemagluttinin (HA or H) so-called because it will cause red blood cells to stick
together, and
neuraminidase (NA or N). The virus is 80-120 nm in diameter and up to 2000 nm long
(variable length).
The third type of projection, the short proteins (unshaded rods) that are not labelled in this
diagram, and which project from between the M1 protein (unshaded circles) capsid, are M2 ions
channels. Two RNPs are visible in the above section. The detailed structure of an RNP is shown
below:
Function of the HA spikes

The haemagluttinin is involved in cell binding and each strain of influenza has one of 16
subtypes of H (designated H1 to H16). Human influenza is characterised by possessing H1, H2
or H3. Three identical copies of the haemagluttinin polypeptide form a single haemagluttinin
spike (it is trimeric, specifically a homotrimer).

HA binds sialic acid-containing receptors on the target cell surface membrane. This is referred to
as docking, binding or adsorption. Sialic acid is a component of carbohydrate chains borne on
the cell surface (the glycocalyx) and also of mucus (mucoproteins).

The virus then tricks the target cell to take it up (by activating the receptor upon binding to it)
and it is taken-up by (coated-pit) endocytosis into a vesicle called a coated vesicle. Acidic
endosomes fuse with this vesicle, as the cell attempts to digest the virus. HA then facilitates the
fusion of the viral and endosome vesicle membranes. This happens when endosomes fuse with
the coated-vesicle, acidifying it. When the pH drops below 6, the HA changes shape, partially
unfolding, which exposes a hydrophobic region (hydrophobic literally means 'water-fearing' and
refers to substances that prefer to dissolve in lipids, rather than water) which attaches to the
endosome membrane, like a grappling hook, then the HA stabilises and refolds, retracting as it
does so and bringing the viral membrane so close to the endosomal membrane that the two
membranes fuse.

Function of the N spikes

The neuraminidase is a sialidase, an enzyme which cuts sialic acid residues from the ends of
carbohydrate chains. This breaks-up the long carbohydrate chains that form the slimey lining of
respiratory airways and makes the mucus of the respiratory tract more watery, which helps the
virus move easily to its target cells (one of the functions of mucus is to entangle foreign
particles, such as viruses, and the N spikes counter this by cutting the entangling threads). The
N spikes help new virus progeny spread from cell to cell. The N spikes will also cleave sialic acid
residues to which HA binds, if the target is inappropriate (such as a mucoprotein in the mucus
layer rather than the cell receptor).

There are also several types of N and the H and N types carried by a particular strain
charcterise the virus. For example, avian flu is H5N1 (avian influenza has one of H1 to H16 and
one of N1 to N9).

Role of the M2 ion channels

These are activated by the low pH when the endosome fuses with the coated-vesicle containing
the virus. They import protons and are involved in triggering uncoating - the disassembly of the
protein capsid which allows the RNPs to enter the host cytosol.

Function of the three polymerase peptides

Once in the cytosol the nucleoprotein which packages the RNA of each RNP, allows movement
of the RNPs into the host cell nucleus. Once inside the nucleus, the three polymerase units (PA,
PB1 and PB2) initiate transcription of the viral RNA. PB2 attaches to the cap present at the head
end (5') of host mRNA, PB1 then cleaves this cap which attaches to PB2 - the virus steals the 5'-
cap off host RNA to make itself look like mRNA! PB1 also adds the usual 3' tail to the end of the
viral RNA, so now it resembles host mRNA ready to be transcribed! PB1 and PA then synthesise
more viral RNA, both mRNA ((+)ssRNA) which is translated by host ribosomes in the cytoplasm
to make viral proteins, and new copies of the viral genome, which remain in the nucleus.

Virus Assembly

Once synthesis results in a high concentration of NP protein in the cytoplasm, viral mRNA
synthesis stops but synthesis of genomic RNA continues - this occurs in the
late phase of
infection and  switches the virus from protein synthesis mode, into assembly mode, in which the
synthesised components are assembled into new virus particles. RNPs are assembled in the
host cell nucleus and are then exported to the cytoplasm.

M protein, HA and N accumulate together in the host cell-surface membrane (after being
processed and delivered there by the rough endoplasmic reticulum and Golgi apparatus of the
host cell). The RNPs assemble here and then the virus buds from the host cell surface,
becoming enclosed in the phospholipd envelope containing the viral protein spikes.

Symptoms of Influenza

  1. Chill, fever (38-39 C)
  2. severe weakness, fatigue, aches and pains in joints and especially in the back and legs,
    sore throat, headache, eye irritation and reddening, reddening of the face and nose.
  3. Abdominal pain in children.
  4. complications that may occur include pneumonia, bronchitis, sinus and ear infections and
    death.

Antivirals

Several chemotherapeutic agents have been developed to combat influenza. These include:

  • M2 inhibitors, block the M2 proton channels with the aim of preventing the viral RNA
    entering the cell cytosol, e.g. amantadine (1-aminoadamantane).
  • Neuraminidase inhibitors, e.g. Tamiflu (Oseltamivir) and relenza (Zanamivir) which
    competes with sialic acid for the enzyme, slowing-down the action of the enzyme.

A vaccine is available, consisting of inactive materials from three different virus strains, which
stimulates the body to produce natural antibodies to influenza.

Influenza replication cycle

The replication or life cycle of influenza is illustrated below. This illustrates many of the features
of a 'typical' animal cell virus, such as:

  1. Adhesion (attachment) of the virus particle (virion) to specific receptors (glycoproteins)
    on the target cell surface.
  2. This adhesion triggers uptake (endocytosis) of the virion by the target cell.
  3. Uncoating and release of the genetic material into the host cell cytoplasm, and
    subsequently nucleus in this case. This genetic material commandeers the machinery of
    the host cell to make more proteins and replicate the viral genome.
  4. Assembly of new virions on the host cell membrane and  their eventual budding from the
    host cell, taking host cell phospholipid, modified with virus proteins, with them as the viral
    envelope.
HIV model (Pov-Ray)
Above: a retrovirus of the lentivirus type, e.g. human immunodeficiency virus (HIV). HIV has
two (+)ssRNA molecules in its genome, represented above as the spirals (red) in the centre of
the elongated inner core (yellow) which are both identical copies of the genome wound with
packaging proteins to form
ribonucleoprotein. The elongated conical core shell (the shape
of which is characteristic of the lentivirus variety of retrovirus, the core being icosahedral in
some retroviruses). HIV is enveloped, that is it is enclosed in a phospholipid bilayer membrane
(cyan) derived from the host cell-surface membrane. This envelope contains glycoprotein
spikes. Just beneath the envelope is a protein shell 9inner shell or matrix) which stabilises the
structure of the envelope.

HIV is well-known as the causative agent of AIDs (Acquired-immunodeficiency syndrome or
acquired immune-deficiency syndrome) in which destruction of key components of the body's
immune system leaves it very susceptible to many other infections. This occurs because HIV
targets certain immune cells, especially T-helper cells (cells that act as alarms and 'officers' for
the immune system - activating and directing other immune cells). HIV will target these cells,
enter them and turn them into virus factories!

Retrovirus Cycle - example HIV

1. Adsorption/adhesion and entry

As usual, the virus must bind to specific receptors on its target cell. These receptors are more-
or-less specific, since viruses infect only one or a few cell types. In the case of HIV, the gp120
spikes bind to CD4 receptors on T-cells (HIV can also infect a few other cell types, such as
macrophages). This is the initial adhesion, which is transient and unstable. This is followed by a
secondary and more stable adhesion between the vrius and a second receptor on the cell
surface (CCR5 or CXCR4). Once stably bound or adsorbed, the lipid envelope of the virus
fuses with the host cell membrane (this fusion is triggered by gp41). The inner core is then
released into the cell.

2. Replication of viral genome

Retroviruses replicate their RNA via a DNA intermediate. They use their RNA as a template to
synthesise first a complementary ssDNA molecule (creating an RNA/DNA hybrid molecule with
the RNA then be degraded leaving ssDNA) and then the ssDNA is used as a template to
generate a dsDNA intermediate. To complete this unusual task, of synthesising dsDNA from a
ssRNA template, HIV carries the viral enzyme
reverse transcriptase (RT) - blue spheres
inside inner core in the diagram above. This enzyme is responsible for:

i) Synthesising the ssDNA complementary strand by using the RNA as a template, that is
working as an RNA-dependent DNA polymerase, resulting in the formation of an RNA/DNA
hybrid molecule.

ii) Digesting away the RNA strand of the RNA/DNA hybrid duplex (
RNAse activity, present as a
separate enzyme or bound to RT and called RNase H) producing ssDNA.

iii) Synthesising the second DNA strand, using the ssDNA as a template, that is working as a
DNA-dependent DNA polymerase, producing dsDNA.

This dsDNA can then be integrated into the host chromosome - it inserts into the DNA of the
host-cell, with the help of the viral enzyme
integrase. In this integrated dsDNA state the virus is
called a
provirus. Note that as this step proceeds efficient gene expression, the virus must
carry integrase, RNase and reverse transcriptase with it. All these enzymes are represented by
the blue spheres inside the viral core.

HIV is unusual in that it can insert its provirus and replicate inside non-dividing cells. To do this
it has to gain access to the nucleus by passing the
nuclear pore complex (NPC). It does this
with the help of at least two viral proteins: MA, which apparently acts as a key granting access
past the NPC and Vpr. replication of the viral RNA takes about 6 hours after cell-infection.

3. Transcription and expression of viral genes

The synthesis of virus proteins only begins efficiently once the provirus is integrated into the
host DNA. The primary transcript (the RNA initially produced by transcription by RNA
polymerase) covers all nine or so genes of the HIV genome and is spliced (cut-up by enzymes)
into more than 30 separate mRNA - 9 genes produce more than 9 proteins, many of which have
multiple functions - a way of minimising on the amount of DNA that must be packaged and
carried by the virion.

The following structural proteins are produced:

  • Gag (p55) - associates with the host cell-surface membrane and recruits two copies of
    the viral RNA genome and other structural proteins for virion assembly and budding from
    the host. It is then cleaved, after the virus has budded and while it is maturing, into the
    following proteins: MA (matrix or p17) which forms the inner protein shell beneath the
    viral envelope which stabilises the virion structure and is also involved in transport of the
    viral RNA to the nucleus, CA (capsid or p24) which forms the conical core or capsid, NC
    (nucleocapsid) which coats the genomic RNA during packaging into the virion and p6, a
    viral protease required for the incorporation of the viral protein Vpr into the virion and
    efficient release of budding virus.

  • Gag-Pol - a protein produced from mRNA that encodes the gag and adjacent pol
    (polymerase) genes. Some 95% of the time, Gap is produced, but 5% of the time, the
    ribosome shifts frame (due to a signal carried on the Gag mRNA and so misses the Gag
    STOP codon and continues onto pol which is encoded on the same mRNA, producing
    Gag-Pol. A viral protease (Pro) then cleaves the Gag from the Pol and cuts up the Pol
    polypeptide into RT (reverse transcriptase, p50), In (integrase, p31), Pro (protease) and
    RNase H (p15, 50% of which remains linked to RT). Pro cleaves Gag and Gag-Pol.
    About 20 Gag are produced to every Gag-Pol.

  • Env - envelope glycoprotein -Env is sent to the host cell's Golgi apparatus where
    carbohydrate chains are added to it (glycosylation) to convert it into a glycoprotein (gp).
    A host cell protease then cleaves Env into gp41 and gp120. These form the envelope
    spikes of HIV: gp41 spans the envelope membrane and the gp120 ligand binds to gp41
    (by a non-covalent bond).

The following regulatory proteins are produced:

  • Tat (transcriptional transactivator) which binds HIV genomic RNA and increases the
    efficiency of its replication 1000-fold.
  • Rev - switches from early to late gene expression.

The following accessory proteins are produced

  • Nef (negative factor) which is produced early and downregulates the CD4 receptor from
    the infected cell (preventing superinfection?). It is also packaged into virions and cleaved
    by viral protease during virion maturation and greatly increases virion infectivity
    (mechanism?).
  • Vpr - incorporated/pacakged into virions; helps the viral DNA gain access to the cell
    nucleus (binds the DNA to the NPC?).
  • Vpu - essential for release of budding virus from the host cell surface; prevents CD$
    inside the cell from binding to Env and blocking its incorporation into developing virions
    by causing the cell to degrade any CD4 bound to Env.
  • Vif - essential for replication; incorporated into the virion; facilitates nucleoprotein
    pacakaging and possibly counters a host cell antivirus mechanism.

Escape from the cell

Gag, gp41 and gp120 gather in patches in the host cell membrane and recruit viral RNA and
other components of the virion. The virus then buds from the host cell, acquiring the lipid
envelope and then completes its maturation.

Evading the host's immune system

HIV has several tricks for evading the defences of its host. Antibodies produced by the host
can, for example, bind to gp120 and prevent new viruses from infecting cells. To combat this,
HIV uses
antigenic variation. In particular, gp120 contains five hypervariable regions, that is
regions whose amino acid sequence and structure differ greatly between one HIV strain and
another. [Recall that
proteins and polypeptides are polymers of amino acids]. The HIV
polymerase (RT + RNase H) also lacks a proof-reading function - it does not check for errors
and so it makes mistakes fairly frequently, causing HIV to mutate extremely rapidly, again
increasing variation. Antigens, e.g. gp120, are exposed regions of foreign proteins and other
large molecules, which the immune system can target and develop antibodies against. By
rapidly varying its antigens, HIV can stay one step ahead of the immune system, changing to a
new form once antibodies are developed, making those antibodies redundant. This helps to
explain why antibodies indicate HIV infection and the possibility of developing full-blown AIDS -
the function of the antibodies is impaired!
Retroviruses
HIV labeled diagram
influenza replication cycle