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Molecular Virology summary book+lectures
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- Wageningen University (WUR)
Molecular Virology (VIR-30306) summary book, lectures and practicals
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Voorbeeld van de inhoud
Molecular VirologyChapter 1- Introduction to Virology
Genetic entities (RNA of DNA), protected by a protein coat and sometimes a lipid membrane, which
upon infection of a suitable host controls the synthesizing system of the cell in such way that new
viruses are produced, often coinciding with visible pathological effects on the cell and the organism:
causal agents of many infectious diseases.
3 main properties:
•infectivity (property to infect a cell, to multiply, and to leave the cell)
•property to stably “survive”in an extracellular state
S•obligatory, intracellular parasites
THE NATURE OF VIRUSES
Virus particles contain:
• A nucleic acid genome (either DNA or RNA) (encodes proteins that enable it to
replicate and be transmitted) (RNA-containing viruses face two related problems as a
result of their RNA genomes: (1) they must synthesize messenger RNAs from an RNA
template, and (2) they must replicate their genome RNA. Most RNA viruses encode
their own RNA-dependent RNA polymerases to carry out both these functions).
• A protein coat (capsid) that encloses the genome
Protein shell (or coat) is built up by smaller subunits: the coat proteins (CPs). For
saving genetic space on the (small) genome, for increasing genetic stability (smaller
gene: lower risk for mutation), for easy (dis)assembly and for constructing a
symmetric virus particle (“virion”)(helps escaping immune system by minimizing the
number of “neutralizing epitops”).
Helical symmetry (no package limitations as particle length is determined by the size
of the genome)
Icosahedral (allows for the lowest-energy configuration of particles interacting isotropically on the surface of a sphere)
(triangulation number)
Complex
• In some cases, a lipid membrane (envelope)
The infectious virus particle is called a virion.
Virus particles are very small: between 20 and 500 nanometers (nm) in diameter.
Viruses are obligatory intracellular parasites (can replicate only within living cells).
Viruses multiply inside cells by expressing and replicating their genomes.
Viruses need the following machinery provided by cells:
• Enzyme systems that synthesize amino acids, nucleotides, carbohydrates, and lipids
• Enzyme systems that generate useable chemical energy in the form of ATP
• Ribosomes, tRNAs, and enzymes used in protein synthesis
• Membranes that concentrate cellular macromolecules, small molecules, and ions
WHY STUDY VIRUSES?
Viruses are important disease-causing agents.
Probably all different forms of life can be infected by viruses.
Viruses can transfer genes between organisms.
Viruses are important players in the regulation of the Earth’s ecology (viruses are the
most abundant form of life on earth).
Viruses can be engineered to prevent and cure disease.
Study of viruses reveals basic mechanisms of gene expression, cell physiology, and
intracellular signaling pathways.
DETECTION AND MEASUREMENT OF VIRUSES
The plaque assay is widely used to measure virus infectivity. Spread bacteria on the
surface of nutrient agar
in a Petri dish and to apply dilutions of a phage suspension. Wherever a phage binds to a
bacterial cell and
,replicates, that cell releases the progeny phage particles, which are then taken up by
neighboring cells and further replicated. After several such cycles all the cells in a circular
area surrounding the original infected cell
are lysed. The lysis area can be seen as a clear “plaque” against the cloudy background
of the uninfected cells. When a plaque assay is used to measure the infectious titer of a
virus suspension, the results are usually
expressed as plaque-forming units (PFU) per mL of suspension. To determine the titer,
the number of plaques
on a plate is multiplied by the factor by which the original virus suspension was diluted
before an aliquot was applied to the plate.
Hemagglutination is a cheap and rapid method for detection of virus particles. Binding
(receptor, virus receptor binding proteins) between an excess of virus particles and an
aliquot of red blood cells forms an interlaced network of cells, held together by virus
particles that form bridges between adjacent cells. These “agglutinated” red blood cells,
when allowed to settle, form a light pink hemispherical shell in the bottom of a tube or
plastic well. In contrast, individual red blood cells slide to the bottom of the tube and form
a compact, dark red pellet. the highest dilution that will agglutinate the aliquot of cells is
considered to have one
hemagglutinating unit (HAU) of virus.
Virus particles can be seen and counted by electron microscopy.
The ratio of physical particles to infectious particles is greater than 1 for many viruses.
This can be due to: not all virus particles being intact, virus particles containing defective
genomes, “empty” capsids that contain no viral genome can be made in large numbers,
cells have antiviral defence mechanisms.
VIRUS REPLICATION CYCLE
1. The virion binds to cell surface receptors (proteins,
carbohydrates or lipids on the cell surface). Many viruses first bind
to a relatively non-specific primary receptor such as a
carbohydrate, and subsequently bind to a specific cell surface
protein that serves as a secondary receptor.
2. The virion or viral genome enters the cell (fusion enveloped
with plasma membrane, vesicles); the viral genome is uncoated.
3. Early viral genes are expressed (Baltimore classification
scheme).
Energetically unlikely that
double-stranded genome is
denatured
No known enzymes that
transcribe ss DNA to RNA
Retroviruses
4. Early viral proteins direct replication of the viral genome. All RNA viruses (except
retroviruses) must synthesize an RNA-dependent RNA polymerase to replicate their
genomes, as this enzyme is
,not present in host cells. The early proteins of many DNA viruses induce the production of
a number of cellular enzymes that are involved in the synthesis of DNA
5. Late viral genes are expressed from newly replicated viral genomes.
6. Late viral proteins package genomes and assemble progeny virus particles.
7. Virions are released from the host cell.
Chapter 3 – Virus Classification
VIRUS CLASSIFICATION
Viruses are classified into related groups based on:
• Genome composition (DNA or RNA, single- or double-stranded)
• Genome topology (linear or circular; single or multiple segments)
• Capsid symmetry
• Presence or absence of an envelope
• Genetic relatedness (nucleotide and amino acid sequence similarity)
• Mechanisms for expressing messenger RNAs and replication of genomes
• Host organisms
Different virus isolates are generally considered to be members of a virus species if they
share a high degree
of nucleic acid sequence identity and most of their proteins have highly similar amino
acid sequences and
common antigenic properties. Virus species are grouped into genera by virtue of shared
characteristics such as genome organization and size, virion structure, and replication
strategies. Genera are in turn grouped into virus families. Members of a virus family share
overall genome organization, virion structure, and replication mechanisms.
THE MAJOR VIRUS GROUPS
Viruses are classified into species, genera, families, and (in some cases) orders, within
the following categories:
• Viruses with single-stranded DNA genomes (small and few genes)
• Viruses with double-stranded DNA genomes (largest known viruses, unfragmented
genomes/single DNA molecule)
• Viruses with positive-strand RNA genomes (most plant viruses and many viruses of vertebrates,
linear genomes, directly infectious, directly translatable)
• Viruses with negative-strand RNA genomes (helical nucleocapsids, linear, some have fragmented
genomes, major infectious disease agents of humans, not infectious, not directly tranlatable)
• Viruses with double-stranded RNA genomes (fragmented genomes and capsids with icosahedral
symmetry)
• Viruses that use a reverse transcriptase (either DNA or RNA genomes, reverse transcriptase in
virion, package 2 copies of genome, vertebrates and plants)
, • Satellite viruses, satellite nucleic acids, (require helper virus to also infect the cell; respectively
encode own capsid proteins or no coding regions/encode only non-capsid proteins) and viroids
(virus-like RNAs that do not code for proteins, but replicate independently of other viruses)
THE EVOLUTIONARY ORIGIN OF VIRUSES
RNA viruses may be relicts of the prebiotic RNA world.
Small DNA viruses could have evolved from escaped, self-replicating fragments of cellular
genome DNA.
Some large DNA viruses could have evolved from degenerate intracellular parasites.
Chapter 10- Cucumber Mosaic Virus
VIRION
Non-enveloped icosahedral capsid (T =3).
Diameter 29 nm.
180 capsid protein subunits (12 pentamers and 20 hexamers). A negatively charged
arginine-rich domain in the
N-terminal region of the coat protein is localized on the internal surface of the virion and
interacts with encapsidated RNA
GENOME
Linear single-stranded RNA, positive sense.
Segmented genome is composed of RNA 1 (~3.4 kb), RNA 2 (~3.1 kb), and RNA 3 (~2.2
kb).
All segments have a 5' cap structure and 3' tRNA-like structure. The tRNA-like structure
may protect viral RNAs from degradation by cellular nucleases, as RNAs covalently bound
to an amino acid are rendered resistant to
ribonucleases that recognize 3' ends of RNAs. Second, the tRNA-like structure may
enhance translation of the
RNA by interacting with cellular proteins involved in translation elongation. Third, tRNA-
like structures may initially reduce synthesis of minus-strand RNA. Because ribosomes
translate the (positive-strand) genome RNA
in the 5'–3' direction, and RNA polymerases proceed in the opposite direction while
synthesizing progeny negative strands, initiation of RNA synthesis during translation of
the same RNA molecule could lead to
collisions between translating ribosomes and transcribing RNA polymerases.
Two subgenomic RNAs: RNA 4 and RNA 4A.
RNAs 1, 2, and 3 are genome RNAs, and simultaneous infection with all three RNAs is
required to establish a productive infection. RNA 1 has a single open reading frame
(protein 1a), RNA 2 has two overlapping reading frames (proteins 2a and 2b), and RNA 3
has two non-overlapping reading frames (protein 3a and coat protein [CP]). The 5'-termini
of the genome and subgenomic RNAs are capped with 7-methylguanosine, as are cellular
messenger RNAs. The 3'-terminal regions of all viral genome RNA segments can fold into
a transfer RNA (tRNA)-like structure. RNA 4 is a subgenomic RNA equivalent to the 3' half
of RNA 3; it encodes the coat protein. RNA 4A is also a subgenomic RNA, equivalent to the
3'-terminal one-quarter of RNA 2; it encodes the 2b protein.
RNAs 1 and 2 are packaged in separate particles, while RNAs 3 and 4 are packaged
together in a single particle.
GENES AND PROTEINS
RNA 1 encodes the 1a protein: a capping enzyme and helicase, and a component of the
replicase (intracellular movement).
RNA 2 encodes the 2a protein: the RNA polymerase component of the replicase, and the
2b protein: a silencing
suppressor (translated from subgenomic RNA 4A) (long distance movement). RNA
silencing is a gene inactivation mechanism mediated by small interfering RNAs
(siRNAs). SiRNAs are generated by the cleavage of
double-stranded RNAs or imperfect hairpin RNAs by the RNase III enzyme, Dicer, or Dicer-
like nuclease. One strand of an siRNA is incorporated into the siRNAinduced silencing
complex (RISC), and this RNA guides
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