Picornaviruses
Introduction:
Picornaviruses are among the most diverse (more than 200 serotypes) and 'oldest' known
viruses (temple record from Egypt ca. 1400 B.C.). FMDV was one of the first viruses
to be recognized - Loeffler and Frosch 1898. Poliomyelitis as a viral disease was
first recognized by Landsteiner and Popper, 1909 (though the virus was not isolated
until the 1930's.
Name: 'Pico (Greek = very small) RNA Viruses'.
Classification:
Originally based on physical properties (particle density & pH-sensitivity)
& serological relatedness, more recently based on nucleotide sequence. The
most recent revision of virus taxonomy has erected 9 genera within the family:
Genome:
The genome consists of one s/s (+)sense RNA molecule of between 7.2kb (HRV14) to 8.5kb (FMDV). A number of features are conserved in all Picornaviruses:
- Genomic RNA is infectious (~1x106-fold less infectious than intact particles, although infectivity is increased if the RNA is introduced into cells by transfection) - CHARACTERISTIC OF (+)SENSE RNA VIRUSES !!!
- There is a long (600-1200 base) untranslated region at the 5' end (important in translation, virulence and possibly encapsidation and a shorter 3' untranslated region (50-100 bases) - important in (-)strand synthesis.
- The 5' UTR contains a 'clover-leaf' secondary structure known as the IRES:
Internal Ribosome Entry Site (see below).
- The rest of the genome encodes a single 'polyprotein' of between 2100-2400 aa's.
- Both ends of the genome are modified, the 5' end by a covalently attached small,
basic protein VPg (~23 AA's), the 3' end by polyadenylation (polyadenylic acid sequences
are not genetically coded, there is a 'polyadenylation signal' upstream of the 3'
end as in eukaryotic mRNAs):
Structure:
The capsid consists of a densely-packed icosahedral arrangement of 60 protomers,
each consisting of 4 polypeptides, VP1, 2, 3 and 4 - all derived from cleavage of
the original protomer VP0, with (pseudo) T=3 packing. The particle is 27-30nm in diameter
(depending on type and degree of desiccation), while the length of the genome (stretched-out)
is ~2,500nm therefore the genome is tightly packed into the capsid, together with
sodium or potassium ions or polyamines (in rhinoviruses) to counteract the negative
charges on the phosphate groups. To view an electron micrograph of negatively-stained
picornavirus particles, click here.
To view a computer generated animation of a picornavirus capsid, click here.
This image is based on the real atomic co-ordinates of rhinovirus 16 and shows a view
inside the capsid. In this video:
- VP1: is in blue
- VP2: is in green
- VP3: is in red
- VP4: is in yellow (only visible on the inside of the particle)
Replication:
We know a great deal about Picornavirus replication due to single-step growth curve
type experiments performed at high multiplicity of infection. Replication occurs entirely
in the cytoplasm - it can occur even in enucleated cells and is not inhibited by actinomycin
D.
Receptors:
The cellular receptors for several different groups of picornaviruses have been identified using a number of different techniques over the last few years:
- Binding competition between different viruses
- MAbs which block virus binding
- Fluorescently labelled virus (Echovirus)
| Virus: |
# Serotypes: |
Receptor: |
Description: |
| Human Rhinovirus |
91 |
ICAM-1 (Intracellular Adhesion Molecule 1) |
Immunoglobulin-like molecule; 5 domains |
| Human Rhinovirus |
10 |
LDLR (Low Density Lipoprotein Receptor) |
| Poliovirus |
3 |
CD155 |
Immunoglobulin-like molecule; 3 domains |
| Coxsackie A |
3 |
ICAM-1 |
| Echo |
2 |
VLA-2 |
Integrin-like molecule |
| Echo |
6 |
DAF (Decay Accelerating Factor) |
??? |
| EMCV |
1 |
VCAM-1 (Vascular Cell Adhesion Molecule) |
??? |
The atomic structure of poliovirus-receptor complex has recently been described:
Belnap
DM et al (Hogle). Three-dimensional structure of poliovirus receptor bound to
poliovirus. PNSA USA 97, 73-78 (2000);
He
Y et al (Rossman). Interaction of the poliovirus receptor with poliovirus. PNAS
USA 97, 79-84 (2000);
Rossmann,
M.G. et al (2000) Cell Recognition and Entry by Rhino- and Enteroviruses. Virology
269: 239-247
The structure of serotype 1 poliovirus bound to CD155 was compared with the
structure of rhinovirus bound to its cellular receptor, ICAM-1. In both cases
the receptor molecule is a long molecule, sticking out from of the surface of
the cell and binding to a "canyon" on the virus particle. However, in the case
of the rhinovirus, ICAM-1 is a long molecule and sticks straight into the canyon,
whereas CD155 lies on the surface of the virus particle along the canyon:
Uncoating:
After adherence to the receptor, the virus can be eluted again, but if this happens,
the particle undergoes conformational changes due to the loss of VP4 and infectivity
is lost - this is also the first stage in uncoating:
Translation:
The kinetics of Picornavirus replication are rapid, the cycle being completed in from 5-10 (typically 8) hours. Genomic RNA is translated directly by polysomes, but ~30 min after infection, cellular protein synthesis declines sharply, almost to zero, this is called 'SHUTOFF' - the primary cause of c.p.e:
| Time after Infection: |
Event: |
| ~1-2h |
Sharp decrease in cellular macromolecular synthesis; margination of chromatin (loss of homogeneous appearance of nucleus) |
| ~2.5-3h |
Start of viral protein synthesis; vaculoation of cytoplasm, beginning close to nucleus & spreading outwards |
| ~3-4h |
Permeabilization of plasma membrane |
| ~4-6h |
Virus assembly in cytoplasm (crystals sometimes visible) |
| ~6-10h |
Cell lysis; release of virus particles |
Shutoff appears to be due to cleavage of the 220kD 'cap-binding complex' (CBC)
involved in binding the m7G cap structure at the 5' end of all eukaryotic mRNAs during
initiation of translation. This is carried out by poliovirus protein 2A.
The 5' UTR contains the IRES: Internal Ribosome Entry Site or 'landing pad'.
Normally, translation is initiated when ribosomes bind to the 5' methylated cap then
scan along the mRNA to find the first AUG initiation codon. The IRES overcomes this
& allows picornavirus RNAs to continue to be translated after degradation of CBC.
The polyprotein is initially cleaved by P2A into P1 & P2P3. Further cleavage events
are carried out by 3C - the main picornavirus protease. All of these cleavages are
highly specific (drug target!):
Read:
Barco,
A. et al. (2000) Poliovirus Protease 3C pro Kills Cells by Apoptosis. Virology
266: 352-360.
Genome Replication:
One of the products made is the virus RNA-dependent RNA polymerase (3D), which copies the genomic RNA to produce a (-)sense strand. This forms the template for (+)strand (genomic) RNA synthesis, which occurs via a multi-stranded replicative intermediate complex (RI). In vitro transcription studies have suggested 2 possible models by which genome replication might occur:
The (-)ve sense cRNA serves as a template for multiple (+)ve sense strands, some of which are translated, others which form vRNA.
Assembly:
RNA is believed(?) to be packaged into preformed capsids, although the molecular interactions between the genome & the capsid responsible for this process are not clear. Empty capsids (defective) are common in all Picornavirus infections. The capsid is assembled by cleavage of the P1 polyprotein precursor into a protomer consisting of VP0,3,1 which join together enclosing the genome:
Maturation:
Maturation (& infectivity) relies on an internal autocatalytic (?) cleavage of VP0 into VP2 + VP4.
Release:
Release (in most cases) on the virus from the cytoplasm occurs when the cell lyses - probably a 'preprogrammed' event which occurs a set time after the cessation of 'housekeeping' macromolecular synthesis at shutoff. (Hepatitis A virus is relatively non-lytic & sets up a more persistent infection).
Enteroviruses
Enterovirus infections are common in humans; seasonal peak in autumn; frequently
undiagnosed:
| Polioviruses |
3 serotypes |
| Coxsackieviruses group A |
23 serotypes |
| Coxsackieviruses group B |
6 serotypes |
| Echoviruses |
31 serotypes |
| Enteroviruses |
38 serotypes |
| Total: |
111 serotypes |
Recently,
a drug has been developed which has activity against enteroviruses and rhinoviruses.
Pleconaril
is a novel drug that inhibits viral replication by blocking viral uncoating,
viral attachment to host cell receptors, and transmission of infectious virions,
with broad-spectrum anti-EV and anti-RV activity and is high bioavailablity
when administered orally.
Polioviruses:
To view a high resolution computer-generated image reconstruction of a poliovirus
particle, click here. Note
the icosahedral symmetry which is clearly visible in this image. These are the prototypic
Picornaviruses; there are 3 distinct serotypes. They cause poliomyelitis (flaccid
muscular paralysis).
As with all the Enteroviruses, they are transmitted by the faecal-oral route.
 |
Primary site of infection is lymphoid tissue associated with the oropharynx
and gut (GALT).
Virus production at this site leads to a transient viraemia, following
which the virus may infect the CNS. This is of interest because of this apparent
'dual tropism' of the virus for two distinct cell types - reflects the distribution
of the poliovirus receptor CD155 on cells lymphoid/ epithelial cells in the gut and
on neurons in the CNS.
Replication of the virus in the CNS occurs in the 'grey matter', particularly
motor neurones in the anterior horns of the spinal cord and brain stem. Distinctive
'plaques' produced in the grey matter are due to lytic replication of the virus &
probably inflammation caused by an over-enthusiastic immune response.
|
~1% of people infected with the most virulent strains experience paralysis (99%
asymptomatic infections). Death is usually due to respiratory failure by paralysis
of the intercostal muscles and diaphragm.
Effective polyvalent vaccines are available against polioviruses - OPV/IPV
. In 1988, the World Health Assembly established the year 2000 for achieving global
poliomyelitis eradication. By 1994, the Americas were certified as polio-free. All
other regions are making steady progress towards this goal:
Coxsackieviruses:
- Algonquin indian name of village in N.Y. where first isolated (Daldorf and Sickles/suckling
mice/1948). Two groups, based on pathology in suckling mice:
- Group A: Cause acute myositis (muscular inflammation) with inflammation
and necrosis. 24 serotypes.
- Group B: Cause degenerative 'plaques' in brain, muscle and pancreas (model
for induced diabetes in mice). 6 serotypes.
- In man, these viruses show a seasonal, epidemic pattern of infection (mostly
sub-clinical), associated with meningitis, paralysis (usually less severe
than acute poliomyelitis), myocarditis, etc. These are common infections world-wide
(inc. UK) - no effective treatment/prophylaxis! Coxsackie A16 causes the common
childhood infection hand-foot-mouth
disease (no, not foot and mouth: hand-foot mouth).
The
Coxsackie B Viruses, by S. Tracy, N.M. Chapman, B.W.J. Mahy (Eds).
This volume reviews current knowledge of the coxsackie B viruses, including chapters
discussing the molecular structure of the virus, the receptor the virus uses to enter
cells, possible effects of nutrition on virus infections and severity of disease,
and more.
(Buy
at Amazon.co.UK)
Echoviruses:
Enteric Cytopathic Human Orphan viruses; not linked
to any human disease (hence 'orphan'). 32 serotypes (echo 10 = reovirus 1; echo
28= HRV1A). Common cause of enteric infections: Outbreak
of Aseptic Meningitis Associated With Multiple Enterovirus Serotypes - Romania,
1999.
'New' Enteroviruses:
Since 1969, 'new' Enteroviruses have been assigned numbers, not names:
| Type: |
Illness: |
| 68 |
Pneumonia |
| 69 |
None (?) |
| 70 |
Acute haemorrhagic conjunctivitis (1969-1974 pandemic) |
| 71 |
Meningitis, Rhombencephalitis |
| 72 |
Hepatitis A virus (now a separate genus: Hepatovirus) |
Cause of 'the common cold' (but not the only one!). ~105 serotypes (hence repeated
infections). Relatively fragile viruses (c.f. Enteroviruses), with optimum growth
temperature of 33°C (URT infection). Extensive human volunteer studies show no
evidence for susceptibility when exposed to cold/wet conditions (!) although general
immune status is probably important. Little c.p.e. Many types grow very poorly in
vitro. Replicate in ferrets - other animal reservoirs? Symptoms due to damage to ciliated
epithelium in URT. Little consequence in itself, but predisposes to secondary bacterial
infections - a major problem in infants and elderly. In addition, a major economic
pest world-wide (lost working days).
No effective prophylaxis or treatment; (Pliny the Younger recommended 'kissing the
hairy muzzle of a mouse') - in spite of extensive molecular knowledge. There is little
or no cross-protection between serotypes. Protection relies on levels of secreted
Ab in URT - may be relatively short-lived (e.g. a few years rather than lifelong).
To view a computer generated animation of a rhinovirus particle, click here.
This image is based on the real atomic co-ordinates of rhinovirus 14. The antigenic
sites on the surface of this particle are highlighted in purple. Note how the distribution
of the antigenic sites on the capsid emphasizes its icosahedral symmetry.
Medscape Article: What's
New With Common Colds? Complications and Management.
This is the group of viruses responsible for foot-and-mouth disease (FMD) - a
major economic pest world-wide, especially in S.America and Australasia. Controlled
largely by vaccination (inactivated vaccine - occasional vaccine-linked outbreaks)
or slaughter of infected animals. They are physically quite distinct from other
Picornaviruses:
- Acid-labile - below pH 7.0.
- Antigenically - 7 serotypes, (A; C; O; SAT1,2,3; Asia-1), location of antigenic sites on capsid quite different from Enteroviruses.
- Genome - Larger than other Picornaviruses, ~ 8.5kb; 5' non-translated region contains poly-C tract of ~100-170nt - function not known (encapsidation?).
To view a high resolution computer-generated image reconstruction of a FMDV particle,
click here.
One serotype. Includes encephalomyocarditis virus (EMCV) (model infection of mice),
mengovirus, Maus-Elberfield virus, Columbia virus - all considered to be strains
of EMCV (really a mouse virus, but can infect man, elephants, squirrels...). Genome
size ~7.8kb; 5' non-translated region contains poly-C tract of ~100-170nt (like
Apthoviruses).