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VIROLOGY - LECTURE 3
DNA VIRUS REPLICATION STRATEGIES
Dr. Margaret Hunt
MBIM 650/720 LECTURES: 52-54
et al., 3rd Ed., Chapter 6, appropriate parts of Chapters 50-52
analysis of the replicative strategies employed by animal DNA
of virus prototypes associated with different DNA virus
Viral genomes contain
of viral genomes
ensures packaging of
genomes into virions
alters the structure
and/or function of the host cell to a greater or lesser degree
The way in which
each virus carries out the above functions
Since a virus is an
intracellular parasite, it has to operate within limits imposed by host
cell, or circumvent them.
DNA VIRUS REPLICATION
The virus needs to
make mRNAs which can be translated into protein by the host cell
The virus needs to
replicate its genome.
Host enzymes for
mRNA synthesis and DNA replication are nuclear (except for those in
mitochondrion) and so, if a virus is to avail itself of these enzymes,
it needs to enter the nucleus.
NUCLEAR DNA VIRUSES
(The name papovavirus
come from papilloma, polyoma, simian vacuolating
are small: 40-60nm
are icosahedral: major capsid protein is VP1, with lesser
amounts of VP2, VP3
have circular, double-stranded DNA is associated with cell
This family contains
two genera: PAPILLOMAVIRUSES AND POLYOMAVIRUSES.
Linda Stannard, University of Cape Town, South Africa.
These viruses are
difficult to grow in culture. They have a different replication strategy
from the polyomaviruses. Papillomaviruses will not be discussed further
this lecture (but see section of DNA
J-Y Sgro, University of Wisconsin. Used with permission
These include SV40, BK,
JC and polyoma viruses. All have a similar strategy for DNA replication.
Depending on the
host cell, they can either transform the cell (go here)
or replicate the virus and lyze the cell.
PENETRATION AND UNCOATING:
proteins interact with cell surface receptors
probably via endocytosis
transported to the nucleus and uncoated
DNA (and associated
histones) enters nucleus, probably through nuclear pore
PRODUCTION OF VIRAL
mRNAS AND PROTEINS:
Gene expression is
divided into early and late phases.
genes encode enzymes
and regulatory proteins needed to start viral replication processes.
genes encode structural
proteins, proteins needed for assembly of the mature virus.
EARLY PHASE OF THE
Note: - - - - indicates regions of the primary transcript which
are removed in the alternatively processed mRNA. Modified from Fiers
et al.,Nature 273:113
The early promoter
is recognized by host RNA polymerase II (SV40 contains a strong enhancer).
transcriptional RNA modification (capping etc.) is carried out by host
transcript (primary transcript) is made and then undergoes
alternative processing, resulting in the mRNAs for the small t and
antigens (these proteins have common amino-terminii but different
The mRNAs are
translated in the cytoplasm.
transcripts which can be processed and code for more than one protein are
seen in several virus families and in the host cell.
LATE PHASE OF THE
By definition the
late phase starts with the onset of viral genome replication.
replication occurs in the nucleus:
T antigen is needed for DNA
replication. This binds to the origin of replication.
uses host cell DNA polymerase, which recognizes the viral origin of
replication if the T antigen is present.
bidirectional (There are two replication
forks per circular DNA genome and replication involves
leading/lagging strands, Okazaki
ligase, etc.). This process of DNA replication is very similar to
that which occurs in the host cell - which is not surprising as the
virus is using mainly host machinery except for the involvement of the
complex with the newly made DNA.
Late gene expression
Late mRNAs are
made after DNA replication (a lot of newly made viral DNA is now
available as template). Early mRNAs are still transcribed, but at a
much lower rate.
T antigen is
involved in controlling the switch on of late transcription and
decreased transcription from early promoter.
VP1, 2, and 3 are
made from same primary transcript which undergoes differential
splicing. This results in the reading frame for VP1 being different
from that for VP2 and VP3:
VP1, 2 and 3 mRNAs
are translated in the cytoplasm, the proteins are transported to
nucleus, and capsids
assemble with DNA (and cell histones) inside capsid.
are released by cell lysis.
TO NOTE ABOUT POLYOMA VIRUS STRATEGY
and late functions
use of the same DNA sequence (alternative splicing,
overlapping reading frames)
protein (T antigen)
genome - so not surprising that virus codes for a very
limited number of proteins
cell provides RNA synthesis machinery, RNA modification
machinery, DNA synthesis machinery, histones for packaging
(modified from Fiers et al., Nature 273:113)
Linda Stannard, University of Cape Town, South Africa.
than papovaviruses (70nm diameter)
icosahedral viruses with fibers
times size of papovavirus genome
DNA is linear, double stranded, associated with virally coded,
basic proteins in virion (unlike papovaviruses, does not use
cell histones to package virion DNA)
Models of the adenovirus virion. A: A 3-dimensional image
reconstruction of the intact adenovirus particle viewed along an icosahedral
3-fold axis (© EMBL Virus Structure Resource). B: A stylized
section of the adenovirus particle based on current understanding of its
polypeptide components and DNA. No real section of the icosahedral virion
would contain all
the components. Virion constituents are designated by
their polypeptide numbers
with the exception of the terminal protein (TP).
Adapted from Fields et al.,
Fundamental Virology (1996).
infect epithelial cells.
The fibers bind to a
cell surface receptor.
The virus is
engulfed by a clathrin-coated
Uncoating occurs in
DNA is released into
the nucleus (probably at a nuclear pore).
Diagrammatic representation of the uptake and uncoating of adenovirus
particles. Adapted from Zinsser Microbiology 20th Ed.
Adenovirus uses host
cell RNA polymerase.
Early mRNAs are
transcribed from scattered regions of both strands.
result in more flexible control.
mRNAs are processed
by host cell capping,
and (sometimes) splicing enzyme systems, they are then exported to the
cytoplasm and translated.
The early proteins
include those which:
are needed for
transcription (E1A protein is needed for transcription of the other
are needed for
adenovirus DNA synthesis (includes DNA polymerase)
alter expression of
host cell genes.
genes whose products:
the host anti-viral response
cell cycle regulation
its own DNA polymerase (which is one of the early proteins).
Adenovirus DNA is
replicated by a strand displacement mechanism .
There are no Okazaki
fragments, both strands are synthesized in a continuous fashion.
Adenovirus DNA replication by a displacement mechanism
cannot initiate de novo, they need a primer. In the case of
adenovirus, the virally coded terminal protein (TP) (not shown in
diagram) acts as a primer. It is thus found covalently linked to the 5'
end of all adenovirus DNA strands.
The way in which
late transcription is switched on is not well understood.
Late mRNAs code
predominantly for structural proteins.
There is ONE major
transcript is processed to generate various monocistronic
There are two types
of cleavage of primary transcript:
i. to generate
various 3' ends which are then polyadenylated
ii. for intron
It is not understood
how this process is controlled such that the correct amounts of each
mRNA are made.
of viral primary transcript
adenovirus particles occurs in the nucleus.
DNA enters the
particles after immature capsids are formed. The capsids then undergo a
The cells lyse and
virions leak out.
proteins are made than are needed, excess structural proteins accumulate
in the nucleus and form inclusion bodies.
TO NOTE ABOUT ADENOVIRUS STRATEGY
are larger and more complex than papovaviruses.
code for their own DNA polymerase and DNA packaging proteins.
although adenoviruses code for their own DNA polymerase, they
use host factors in addition to viral proteins for DNA
replication, and they use host RNA polymerase and RNA
modification systems and so nucleic acid synthesis needs to be
in the nucleus.
OF HERPES VIRUSES
virions (180-200nm) than adenoviruses
genome (90-145x106) than adenoviruses
icosahedral virus (this means that lipid solvents readily
inactivate these viruses)
Herpes virus structure
Herpes simplex virus © Dr
Linda M Stannard, University of Cape Town, South Africa, 1995 (used
including herpes simplex virus, can fuse directly with the cell plasma
membrane (results in partial uncoating).
Herpes simplex virus adsorbing to the plasma membrane © Dr
Dennis Kunkel, University of Hawaii. Used with permission
fusion with plasma membrane:
i) Since the
fusion protein is active at physiological pH, if it is inserted into
the host cell membrane during the virus growth cycle, that cell can
potentially fuse with other cells and form syncytia.
ii) In addition,
viral membrane leaves "footprint" - possible clue that the
cell is infected
Fusion of membrane-bound virus with the plasma membrane
transported towards the nucleus and the DNA passes into the
nucleus (probably via nuclear pores).
Expression of immediate early, early and late genes of herpesviruses
(the mRNAs made during this phase are the alpha and beta mRNAs)
uses host RNA polymerase. (A virion protein enters the nucleus upon
infection and is important as part of the transcription factor complex
recognized by the host RNA polymerase.)
It uses host mRNA
are transcribed. These are exported to the cytoplasm and translated into
translated in the cytoplasm are transported into nucleus.
enable the ß-promoters to be used by the host RNA polymerase.
transcribed the by host RNA polymerase.
still "early" since they are transcribed prior to DNA
synthesis. Sometimes therefore, a-genes are called "immediate
early" and ß-genes are called "early").
involved in gene expression regulation (they decrease a-gene expression
and are needed for ?-gene expression) and in various aspects of DNA
synthesis: e.g. herpes ß-genes code for:
SINCE THESE BETA
PROTEINS ARE VIRALLY-CODED AND NOT HOST-CODED ENZYMES, THEY ARE
POTENTIALLY WEAK LINKS IN THE VIRUS LIFE CYCLE AND THUS PROMISING TARGETS
FOR VIRAL CHEMOTHERAPY
DNA circularizes in
cell, replicates via a rolling circle mechanism, forming tandem repeats
which are then cleaved. A simplified scheme is shown here:
Rolling circle DNA replication
have a complicated genome structure in which two parts of the genome can
invert relative to each other, e.g. herpes simplex virus, others do not.
The significance of this is unclear.
Herpes: Possible genomic structures
occurs after DNA replication (by definition).
?-mRNAs are made,
these are translated in the cytoplasm.
There is decreased
expression of ß-genes in the late stage. This is probably due to
down-regulation of transcription of ß-genes by one of the
there is no apparent organization of the genome into blocks for either
early or late transcription.
Stages in the exocytosis
of herpes virus from the nucleus, in which the virus core is assembled, to
the plasma membrane
Assembly occurs in
the nucleus. A capsid is formed and the DNA enters the capsid.
The capsids bud
through localized areas of the inner nuclear membrane which have viral
membrane proteins inserted into them. (These areas have tegument
proteins associated with the inner face of the inner nuclear membrane).
In some undefined
way, virions are released to the environment, the diagram above
indicates one possible route.
proteins accumulate in nucleus, often form inclusion bodies (part of the
CYTOPLASMIC DNA VIRUSES
Negative stain and thin section of pox viruses © F.
There are several
reasons why poxviruses are of importance:
poxviruses are of historic note such as smallpox and vaccinia, cow
pox, which was used in the smallpox vaccine (go here)
Boy with smallpox CDC/Cheryl Tryon firstname.lastname@example.org
(b) Pox viruses are
used in new techniques of vaccine development (such as
(c) Some members of this family infect man (molluscum contagium, orf,
Transmission electron micrograph of poxvirus of molluscum contagiosum
genome, double-stranded DNA 130 - 240x106 mol. wt.
in the cytoplasm. This means that they must provide their own mRNA and
DNA synthetic machinery.
Vaccinia is the most
intensively studied member of the poxvirus family.
The virus binds to
cell surface receptors.
It enters cells via clathrin-coated pits or by direct fusion of the
virus with the plasma membrane.
The virus is then released into the cytoplasm, minus its membrane.
After the initial
phase of uncoating has occurred, the virus can make a limited number of
mRNAs (the immediate early mRNAs).
To do this, the
poxvirus needs a DNA-dependent RNA polymerase. Host RNA polymerase is
in the cell nucleus and so this explains why poxviruses use a virally-coded
DNA-dependent RNA polymerase to make their RNAs. Since this enzyme
is needed immediately upon infection, it must be brought into the
infected cell with the vaccinia DNA, it is thus present in virions.
vaccinia DNA which has had all the protein removed is thus not
infectious, since it will have no RNA polymerase associated with it, and
nothing can happen in the vaccinia life cycle without the vaccinia RNA
Poxvirus mRNAs are
capped, methylated and polyadenylated just like standard eucaryotic
mRNAs, but host cell mRNAs are modified in the nucleus and vaccinia
replicates in the cytoplasm. Since Vaccinia is cytoplasmic, these
modifications must be carried out by virally-coded enzymes. The
modifying enzymes are packaged in virions and thus those mRNAs made
immediately upon infection can be modified.
So far, no spliced
mRNAs have been reported for vaccinia (this is not surprising since it
replicates in cytoplasm and host splicing enzymes are in the nucleus).
One of the immediate
early mRNA translation products is an uncoating enzyme. This allows
further uncoating of the vaccinia DNA and more genes can now be
transcribed - the early genes are now all expressed. Poxviruses are
exceptional in that they code for an uncoating protein which has to be
made in the newly infected cell before uncoating can be completed.
"factories" are seen in the cytoplasm - sites of vaccinia virus
The early proteins are
involved in DNA replication, RNA transcription, RNA modification and
uncoating. They also include a few structural proteins.
Uses an unusual
mechanism which will not be dealt with here.
Late transcription and
This is a complex
Some late proteins
are made throughout the late phase, but others only at the beginning
of late phase.
proteins are not synthesized once DNA replication commences while
other early proteins are made during late as well as early phases.
Thus, there is
complex control of which proteins are made by vaccinia and when they
are made. This means that there are controls other than just
early/late controls. (This is a very large virus, thus the complexity
is not surprising.)
Assembly occurs in
"factories" in the cytoplasm.
The new, immature
virus particles acquire a membrane while in the cytoplasm - the exact
mechanism is not fully understood but it seems that the virus gets
"wrapped" by cellular membranes.
There is a gradual
maturation of enveloped particles.
The virus is usually
released by host cell disintegration, but some may get out by budding
through membranes (in which case they have an extra membrane). Both
forms appear to be infectious.
The exact mechanism
by which the virus gets out of infected cells may depend on host cell
Possible scheme for the formation of infectious pox virions. The virus
core becomes wrapped in cytoplasmic membrane and may escape when t
cell is lyzed. Some other membrane-bound virions may bud through other
membranes, in which case they have two membranes. In either case, the
are infectious. Adapted from Baron, S. Ed. Medical
Microbiology 4th Edition. 1996.