VIROLOGY - LECTURE TWO
BASIC VIROLOGY: REPLICATION OF VIRUSES
Dr. Margaret Hunt
MBIM 650/720 LECTURE 51
READING: Murray et
al., Microbiology, 3rd. Ed., Chapter 7
overall view of the replication cycle of viruses
INVOLVED IN REPLICATION
Virus attaches to the
cell surface. Attachment is via ionic interactions which are
protein recognizes specific receptors on the cell surface (These may be
protein or carbohydrate or lipid components of the cell surface).
Cells without the
appropriate receptors are not susceptible to the virus.
(Virus enters the cell)
(A) Entry by fusing
with the plasma membrane. Some enveloped viruses fuse directly with
the plasma membrane. Thus, the internal components of the virion are
immediately delivered to the cytoplasm of the cell.
(B) Entry via
endosomes at the cell surface
Some enveloped viruses require an acid pH for fusion to occur and are
unable to fuse directly with the plasma membrane. These viruses are taken
up by invagination of clathrin coated pits into endosomes. As the
endosomes become acidified, the latent fusion activity of the virus
proteins becomes activated by the fall in pH and the virion membrane fuses
with the endosome membrane. This results in delivery of the internal
components of the virus to the cytoplasm of the cell
may cross the plasma membrane directly or may be taken up via clathrin-coated
pits into endosomes. They then cross (or destroy) the endosomal membrane.
Nucleic acid has to be
sufficiently uncoated that virus replication can begin at this stage. When
the nucleic acid is uncoated, infectious virus particles cannot be
recovered from the cell - this is the start of the ECLIPSE phase
- which lasts until new infectious virions are made.
4. SYNTHESIS OF VIRAL
NUCLEIC ACID AND PROTEIN
A lot of strategies
are used, some will be discussed later.
New virus particles
are assembled. After assembly they may undergo a maturation process.
Virions may be
released due to cell lysis, or, if enveloped, may bud from cell. Budding
viruses do not necessarily kill the cell. Thus, some budding viruses may
be able to set up persistence.
Transmission electron micrograph of HIV-1, budding and free
HIV budding from human lymph tissue (TEM x133,335) Copyright Dr
Dennis Kunkel, University of Hawaii (used with permission)
All proteins in a
mature virus particle are said to be structural proteins - even if they
make no contribution to the morphology or rigidity of the virion -
non-structural proteins are those viral proteins found in the cell but not
packaged into the virion.
Not all released virus
particles are infectious. The ratio of infectious to non-infectious
particles varies with the virus and with growth conditions.
Some methods (e.g.
electron-microscopy) enable every virion to be counted but are not
informative about infectivity. Other methods (e.g. hemagglutination) are a
less sensitive measure of how much virus is present, but again are not
informative about infectivity. Other methods, e.g. plaque assay, measure
the number of infectious virus particles.
EFFECT OF VIRUSES ON
HOST MACROMOLECULAR SYNTHESIS
Many viruses inhibit
host RNA, DNA or protein synthesis (or any combination of these). The
mechanisms by which the virus does this vary widely.
The presence of the virus often gives rise to morphological changes in the
host cell. Any detectable changes in the host cell due to infection are
known as a cytopathic effect. Cytopathic effects (CPE) may consist of cell
rounding, disorientation, swelling or shrinking, death, detachment from
the surface, etc.
Many viruses induce
apoptosis (programmed cell death) in infected cells. This can be an
important part of the host cell defense against a virus - cell death
before the completion of the viral replication cycle may limit the number
of progeny and the spread of infection. (Some viruses delay or prevent
apoptosis - thus giving themselves a chance to replicate more virions.)
Some viruses affect
the regulation of expression of the host cell genes which this can have
important results both for the virus's ability to grow, and in terms of
the effect on the host cell.
The CPE effect can be
used to quantitate infectious virus particles by the plaque-forming
Cells are grown until
they form a monolayer of cells covering a plastic bottle or dish. They are
then infected with the virus. The liquid growth medium is replaced with a
semi-solid one so that any virus particles produced as the result of an
infection cannot move far from the site of their production. A plaque
is produced when a virus particle infects a cell, replicates, and then
kills that cell. Surrounding cells are infected by the newly replicated
virus and they too are killed. This process may repeat several times. The
cells are then stained with a dye which stains only living cells. The dead
cells in the plaque do not stain and appear as unstained areas on a
colored background. However viruses which do not kill cells may not
A plaque assay. Serial dilutions of virus have been plated on confluent
monolayer cultures of cells. The cells are stained after a period of time
in which a single virus infects a cell, produces new virus particles and
infects surrounding cells. The white areas show areas of the culture in
which the cells have been killed. Each "plaque" is the result of
the presence of one original infectious virus particle.
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