Transcript Expression and Replication of the Viral Genome in Eukaryotic Cells: The DNA Viruses

Expression and Replication of
the Viral Genome in
Eukaryotic Cells: The DNA
Viruses
(How do DNA viruses express
their genomes in Eukaryotic
cells?)
Eukaryotic DNA Viruses
 The DNA viruses have a larger range in size
than do the RNA viruses.
 Since their genome is of the same molecular
type (DNA) as their host’s genome, they can
use the host cellular machinery for
replication, transcription and translation.
 If they are dependent upon host cell enzymes
for replication they need to get to the host
cell nucleus, and problems that they may
encounter in replication are:
Eukaryotic DNA Viruses
 The host cells must be in the S phase of the cell
cycle when the enzymes for DNA replication are
made and would be available for the virus to use.
To overcome this problem:
 Small DNA viruses only infect cells that have naturally
entered the S phase of the cell cycle.
 Other viruses have a way to force the host cells into the
S phase of the cell cycle.
 Larger viruses simply encode their own enzymes for this
process. That way they are not dependent on the host
cell enzymes.
 If these viruses also bring in the enzymes for transcription,
they may replicate entirely within the cytoplasm since they
don’t need replication or transcription enzymes/proteins
present in the host cell nucleus.
Eukaryotic DNA Viruses
 If the genome is linear, DNA synthesis can’t
occur at the 5’ ends of the template
(lagging strand synthesis). This results in 3’
overhangs. To overcome this problem:
 The virus may use protein primers
 The virus may form concatamers
 The virus may resort to the use of reverse transcription.
 Expression
 Early genes encode nonstructural regulatory
proteins for DNA replication
 Late genes are expressed after DNA replication
and they encode the structural components of the
virus.
Eukaryotic DNA Viruses
 Outcomes when a DNA virus infects a cell
 Productive, acutely cytopathic infection – leads to
the manufacture and release of new virions and,
in the process, the infected cell dies.
 Lysis
 Apoptosis
 Persistent infection - the virus remains present for
long periods of time without killing or seriously
impairing cell function. A balance is struck
between the virus and the host. This can also
occur with RNA viruses, but it occurs more often
with DNA viruses. There are two types of
persistent infection:
Eukaryotic DNA Viruses
 Chronic infection –can always recover infectious
virus from the host
 Due to specific virus-host interactions
 Due to antibody or interferon production that limits,
but does not completely inhibit virus production
 Due to production of defective-interfering particles
 Due to a combination of all three of the above
 Latent infection – the viral genome persists, but
infectious virus can only be recovered during
reactivation periods (more on this later on)
Eukaryotic DNA Viruses
 Transformation – viral infection changes the cellcell communication and growth regulation in the
host cell which then takes on the characteristics of
a tumor cell (more about this later on).
 The viral genome or part of it is retained, but infectious
virus is usually not produced.
 In general, for DNA viruses that are capable of causing
transformation, in a permissive host there is a productive
infection and the host cell dies rather than being
transformed.
 In a nonpermissive host transformation may occur.
Eukaryotic DNA Viruses
 Double stranded DNA viruses
 Papovaviruses – includes 2 subfamilies –
polyomavirinae (polyoma and SV40), and
papillomavirinae
 They are small icosahedral viruses
 They have a circular DS DNA genome that encodes 5-7
proteins. We will look at SV40 as an example:
 The DNA is complexed with histones and encodes 6
proteins, 2 nonstructional proteins (early genes = large T
Ag and small T Ag) and 4 structural proteins (VP1,VP2,
VP3, and agnoprotein)
SV40
 Transcription
 Uses host RNA polymerase II
 Early genes encode the T Ags. that are
expressed from differentially spliced mRNAs
 Late genes encode VP1, VP2, VP3, and the
agnoprotein which are all expressed from 2
differentially spliced mRNAs.
 VP2 and VP3 are expressed from the same
mRNA using different start codons.
 The agnoprotein is expressed from the same
mRNA as VP1 using a different start codon.
SV40 Virus
SV40 virus
Early gene expression
Late gene expression
SV40 Virus
 The early promoter contains a TATA box and is
activated by host cell transcription factors and low
concentrations of one of its products, the large T
antigen
 The late promoter does not contain a TATA box. The
shift from early to late gene expression occurs after
DNA synthesis has begun.
 The late promoter region has multiple binding sites for
a cellular repressor, Ibp.
 Once viral replication begins, the concentration of the
late promoter becomes sufficiently high such that not
all Ibp binding sites are occupied and the promoter
becomes accessible to cellular transcription factors.
 The large T Ag binds to the promoter to help activate
late gene transcription and to suppress early gene
transcription.
SV40 Virus – late gene
expression
SV40
 Replication - The Large T Ag plays a role and has:
 ATPase activity
 Binding to DNA origin of replication
 Can serve as a DNA unwinding helicase.
 It opens the DNA at the origin of replication to let in a
host cell primase complex to initiate bidirectional DNA
replication.
 This results in a pair of relaxed DS molecules that get
supercoiled by host topoisomerase II
 Gene activator activity for genes in the host cell that are
involved in controlling the host cell cycle.
 Cellular DNA synthesis is induced so the the enzymes that
the virus requires to replicate its genome are available.
 Large T Ags presence in tissue culture cells can result in
immortalization of the cells ( cells are permanently going
through the cell cycle, therefore they can be passaged
again and again)
 Replication of the circular genome involves a theta form
SV40 T Ag activities
SV40 genome replication
Eukaryotic DS DNA Viruses
 Adenovirus
 Has a naked, icosahedral capsid
 Has a linear, double-stranded DNA genome with
 Inverted terminal redundancies
 A 55 kd terminal protein attached covalently to the 5’ ends
 Transcription
 Both strands serve as templates for transcription by the
host cell DNA dependent RNA polymerase II. Therefore,
the strands are called the right and the left strand to
indicate the direction of transcription.
 Immediate early gene – expression of E1A, the immediate
early gene, is needed for the expression of the remaining
early genes
 Early genes – use 5 different promoters with each product
being spliced in at least three different ways. The early
gene products are involved in the regulation of viral and
cellular synthetic activities (S phase activation)
 Late genes are expressed after the onset of DNA synthesis
and from a single promoter with a complex number of
splicing reactions of the product.
Adenovirus
Adenovirus
Adenovirus
 Replication – how can the virus replicate the ends of its
linear genome?
 There is a terminal protein (TP) at the 5’ end of the
genome that is involved as is a viral DNA polymerase, a
viral single stranded binding protein, and two host cell
transcription factors that the virus borrows to assist in its
replication.
 All viral DNA synthesis is continuous.
 The host cell factors bind to and alter the conformation of
the 3’ end of the DNA to promote binding of the viral DNA
polymerase - TP precursor(80 kd protein) complex.
 The 80 kd TP precursor serves as a primer for DNA
synthesis. Elongation from an OH a serine side chain
causes displacement of the corresponding genomic DNA
strand.
 Both strands may initiate synthesis at the same time or
only one strand may initiate synthesis.
 Both strands use the 80 kd terminal protein precursor as a
primer. It is cleaved to the 55 kd terminal protein during
viral maturation.
Adenovirus
Adenovirus genome replication
Eukaryotic DS DNA Viruses
 Herpesviruses
 Have an envelope and an icosahedral capsid
 Between the capsid and the envelope is a matrix called
the tegument
 The genome is a linear, double stranded DNA with nicks
or gaps
 The  herpes viruses are neurotropic
 The  and  herpesviruses are lymphotropic
 The  herpesviruses have been linked to human cancers
(EBV and Kaposi sarcoma-associated virus)
 All form latent infections after the initial primary infection
 Include HSV 1 and 2, varicella-zoster, EBV, Kaposi
sarcoma-associated virus, and CMV
Herpesviruses
 Transcription
 Some use differential splicing and/or alternate promoters
 HSV-1 uses neither, but has a complex way of regulating
the timing of gene expression
 Immediate early genes () – products are involved in
regulating the expression of all three sets of genes (,
, and )
 Early genes () – products are involved in nucleic acid
metabolism and DNA synthesis (they duplicate cellular
genes)
 Late genes () - products are the structural proteins
 The control of all three sets of genes is by feedback
loops
HSV-1
 A  gene product ( trans induction factor or VP16)
which is a virion tegument protein, enters the host
nucleus with the genome and activates  gene
transcription.
HSV-1
 Replication
 Many  gene products are involved in DNA
replication – only 7 encode required functions
 Host cells do not need to be in the S phase of the
cell cycle since the virus provides its own replication
machinery
 A model involving circularization of the linear
genome due to single nucleotides extensions that are
complementary at each 3’ end
 Initial circle amplification
 Rolling circle replication to create concatamers
 Cleavage of concatamers during incorporation into
virions
HSV-1 genome replication
5’ end
’
Herpesvirus – proposed model
for DNA replication
Eukaryotic DS DNA Viruses
 Poxviruses
 Are the largest of all known viruses
 Have a core plus 2 lateral bodies and a
lipoprotein coat plus an envelope
 Has a double stranded linear DNA genome with
closed ends and termini with inverted repeats
 Carry all their own enzymes for replication and
transcription, both of which occur in the
cytoplasm
Poxvirus genome
Poxviruses
 Transcription
 Begins as soon as the viral core is released into the
cytoplasm.
 All enzymes required to produce what looks like eukaryotic
mRNA are part of the virus itself.
 Half of the genome is expressed from the early gene
promoters.
 Late gene transcription occurs during and after DNA
replication and is controlled by regulatory proteins and the
configuration of the newly made DNA.
 After DNA replication begins, early mRNAs are not
transcribed as efficiently, therefore less early mRNA is
made. Those that are made are also degraded more
rapidly.
Poxviruses
 Replication
 Concatamers that are head to head and tail to tail rather
than head to tail are formed
 A nick is made near 1 terminal repeat. This creates a free
3’ OH that serves as a primer for synthesis.
 The sequence now available to serve as template contains
a set of bases that are self-complementary
 The newly added bases on the 3’ end are also selfcomplementary so they can fold back on themselves to
begin a process of displacement synthesis
 The displaced DNA serves as a template to form a 2
genome long concatamer.
 The growing point doubles back on itself to use the newly
replicated DNA as a template to create 4 genome length
concatamers.
 Eventually staggered cuts and ligation create 4 genomes.
Poxviruses
Poxviruses
 Replication can occur from both ends at
the same time
Eukaryotic DNA Viruses
 Single stranded DNA viruses
 Parvoviridae
 Naked and small with a linear, SS DNA genome
 Have no means of forcing the host into the S phase of
the cell cycle
 Those that infect mammalian or avian cells have (-)
strand DNA. Others package either (+) or (-) strand
DNA.
 There are three genera
Parvoviridae
 Densoviruses – reproduce in insect cells
 Parvoviruses – reproduce in suitable mammalian hosts
 Dependoviruses – are replication defective. An
example is the adeno associated viruses (AAV). They
require that the host be infected with another virus to
provide helper functions necessary for replication and
they can package either the (+) or the (-) DNA strand.
 Autonomous viruses have all the information
necessary to reproduce in a suitable host cell and they
package (-) sense DNA strands as their genome.
 The genomes of the parvoviruses contain selfcomplementary sequences at the ends that form
hairpin loops. The sequences at the two ends are the
same fro the dependoviruses, but different from each
other for the autonomous viruses.
Parvovirus
Parvovirus
 Expression of the parvovirus genome involves
the use of three different promoters (two for the
early, nonstructural and regulatory genes, and
one for the late, structural genes), use of
differential splicing, and alternative AUGs
Adeno-associated Virus
Replication
 Replication of parvoviruses is very complicated and
involves self-priming because of the self-complementary
sequences at each end. It involves SS displacement
synthesis leading to concatamer formation.
 The replication defective AAV have self-complementary,
inverted repeats at their 5’ and 3’ ends.
 The hairpin loop in the 3’ terminus serves as a primer
for elongation down the entire length of the genome
 A nick in the original strand permits the hairpin to
straighten out and thus serve as template for
elongation of the newly created 3’ end
 Note that each of the resulting DNA molecules is a
hybrid of new and old DNA
 Note that the sequence of some of the bases within
the terminal repeat has been reversed.
Adeno-associated Virus Replication
Autonomous parvovirus genome
replication
Eukaryotic DNA Viruses
 Hepadenaviruses
 Are very small with a capsid about the size of
parvovirus
 Have an envelope with glycoprotein spikes
 Genome is partially SS, circular, nicked DNA
 The longer strand is the (-) strand.
 It has a protein attached to the 5’ end and it serves as a
template for transcription of all viral mRNAs.
 The shorter strand is of varying length.
 It has a 19 basepair RNA attached to its 5’end.
Hepadenaviruses
 The two strands basepair in an overlapping
fashion with breaks in both strands.
 Note that there is a pair of direct repeated
sequences, DR1 and DR2.
Hepadenaviruses
 We will look at the human hepatitis B virus
 Immediately upon entering a host cell, the genome is
trasnsported to the nucleus and the shorter strand
elongates to the length of the longer strand, both
strands become unblocked at their 5’ ends, and the
genome is converted to a covalently closed circular DNA.
 Transcription uses the host RNA polymerase II and
occurs from 4 viral promoters.
 Using these promoters, 1 genomic and 3 subgenomic
mRNAs are made.
HBV
 The 4 mRNAs encode seven proteins.
 The mRNAs all start at different 5’ sites, but
have the same 3’ terminus
 The largest is longer than the entire genome –
the promoter is upstream of the 3’ cleavage/poly
A site, but the poly A site is not used until the
second time it is encountered .
 Two of the mRNAs (for the genomic and S
promoters) show heterogenous 5’ ends.
 Expression of P from the genomic mRNA may
occur from ribosome slippage during translation.
HBV
HBV
 Replication
 Uses an RNA intermediate.
 Uses the 2 direct repeat sequences, DR1 and DR2
 Begins with synthesis of a pregenomic RNA
equivalent to the longest mRNA.
HBV
 Reverse transcriptase synthesizes a (-) DNA strand
using the pregenomic RNA as a template.
 Synthesis begins at the 3’ DR1
 Synthesis is primed by the viral DNA polymerase
acting as a protein primer attached to the 5’ end.
 All the pregenomic RNA except the DR1 region is
degraded by the RNAse H activity of the RT.
 Synthesis of the (+) strand requires a jumping
reaction in which the DR1 at the 5’ end of the
(+) strand basepairs to the DR2 at the 5’ end of
the (-) strand.
HBV
HBV
 Circularization of the negative strand allows the viral
polymerase to synthesize the (+) strand using the
RNA as a primer.
 Assembly occurs during these steps. DNA synthesis
ceases when the particle leaves the cell. Therefore
different lengths of the (+) strand are made.
HBV - summary