7

Virus genome replication

At a glance
Baltimore
Class

DdDp/DdDp 1
I-II DNA DNA

RdRp
III-V RNA RNA

RdDp DdRp
VI RNA DNA RNA

DdRp RdDp
VII DNA RNA DNA

Virus enzymes: DdDp = DNA-dependent DNA polymerase

RdRp = RNA-dependent RNA polymerase
RdDp = RNA-dependent DNA polymerase (reverse transcriptase)

Cell enzymes: DdDp = DNA-dependent DNA polymerase

DdRp = DNA-dependent RNA polymerase (RNA pol II)

1
Some dsDNA viruses use a cell DdDp, some encode their own.

Virology: Principles and Applications John B. Carter and Venetia A. Saunders

2007 John Wiley & Sons, Ltd ISBNs: 978-0-470-02386-0 (HB); 978-0-470-02387-7 (PB)
86 VIRUS GENOME REPLICATION

At a glance (continued)
Locations of virus genome replication in eukaryotic cells

CYTOPLASM some dsDNA viruses

dsRNA viruses
(+) RNA viruses
(−) RNA viruses (non-segmented genomes)
retroviruses & pararetroviruses (RNA → DNA)

NUCLEUS

NUCLEUS some dsDNA viruses

ssDNA viruses
(−) RNA viruses (segmented genomes)
retroviruses & pararetroviruses (DNA → RNA)

Primers
3’
DNA OH (self-priming)

RNA 3’OH

protein OH

7.1 Overview of virus DNA intermediate. The various replication modes of

genome replication
In this chapter we consider the fifth step of our
generalized replication cycle: genome replication. The
genome of the infecting virus is replicated so that viral
transcription can be amplified and to provide copies of
the genome for progeny virions.
Generally, DNA viruses copy their genomes directly
to DNA and RNA viruses copy their genomes directly
to RNA. There are, however, some DNA viruses that
replicate their genomes via an RNA intermediate and
some RNA viruses that replicate their genomes via a
virus genomes are summarized in Figure 7.1.
Single-stranded genomes are designated as plus or
minus depending on their relationship to the virus
mRNA. Plus strand genomes have the same sequence as
the mRNA (except that in DNA thymine replaces
uracil), while minus-strand genomes have the sequence
complementary to the mRNA. Single-stranded DNA is
converted to dsDNA prior to copying.
There are two classes of viruses with (+) RNA
genomes (Figure 7.1). Class IV viruses copy their (+)
RNA genomes via a (−) RNA intermediate, while Class
VI viruses replicate via a DNA intermediate. The
 OVERVIEW OF VIRUS GENOME REPLICATION 87

Class

dsDNA dsDNA
DNA Viruses I

II (+) DNA (+) DNA

or
or + dsDNA
(–) DNA – (–) DNA

+ dsRNA dsRNA
RNA Viruses III +
– –
or

(+) RNA dsRNA

+
–

(+) RNA (–) RNA (+) RNA

IV

V (–) RNA (+) RNA (–) RNA

(+) RNA
Reverse-Transcribing VI
Viruses

(–) DNA dsDNA (+) RNA

+
–

+ dsDNA
VII –

(+) RNA (–) DNA + dsDNA

–

Figure 7.1 Replication of virus genomes in the seven Baltimore Classes. (+) RNA and (+) DNA have the same
sequence as the mRNA (except that in DNA thymine replaces uracil). (−) RNA and (−) DNA have the sequence
complementary to the mRNA (except that in DNA thymine replaces uracil). (+) and (−) strands are not indicated
for the dsDNA of the Class I viruses as the genomes of most of these viruses have ORFs in both directions. (+)
and (−) strands are indicated for the ssDNA of the Class II viruses. Most of these viruses have either a (+) or a
(−) strand genome. Some ssDNA viruses and some ssRNA viruses have ambisense genomes.
88 VIRUS GENOME REPLICATION
synthesis of DNA from an RNA template (reverse tran-
scription) is also a characteristic of Class VII viruses.
In this chapter we shall look at some general aspects
of virus genome replication, and then we shall give
individual attention to replication of the genomes of the
DNA viruses, the RNA viruses and the reverse
transcribing viruses.
7.2 Locations of virus genome
replication in eukaryotic cells
As we saw in Chapter 5, when viruses infect eukaryotic
cells the genomes of some are delivered to the
cytoplasm and some are conveyed to the nucleus. The
destination of a virus genome, and hence the location in
which it is replicated, varies with the type of genome
(Table 7.1).
The genomes of most DNA viruses are replicated in
the nucleus, but those of some dsDNA viruses are
replicated in the cytoplasm. The genomes of most RNA
viruses are replicated in the cytoplasm, but those of the
minus-strand RNA viruses with segmented genomes are
replicated in the nucleus. The retroviruses and
pararetroviruses are special cases: each replicates RNA
to DNA in the cytoplasm and DNA to RNA in the
nucleus.
7.3 Initiation of genome replication
Each virus genome has a specific sequence where
nucleic acid replication is initiated. This sequence is
recognized by the proteins that initiate replication.
Table 7.1 Locations of virus genome replication in eukaryotic cells
Virus genome
dsDNA
ssDNA
dsRNA
(+) RNA
(−) RNA (non-segmented genome)
(−) RNA (segmented genome)
Retroviruses [(+) RNA]
Pararetroviruses [dsDNA]
3’
OH
Figure 7.2 3 end of a ssDNA where self-priming
of DNA synthesis can occur.
Nucleic acid replication requires priming, which is
the first reaction of a nucleotide with an –OH group on a
molecule at the initiation site. Replication of the
genomes of many RNA viruses (including rotaviruses,
and rhabdoviruses) initiates when the first nucleotide of
the new strand base pairs with a nucleotide in the viral
RNA. The initial nucleotide effectively acts as a primer
for RNA replication when its 3 –OH group becomes
linked to the second nucleotide.
Some ssDNA viruses, such as parvoviruses, use self-
priming. At the 3 end of the DNA there are regions with
complementary sequences that can base pair (Figure
7.2). The –OH group of the nucleotide at the 3 end
forms a linkage with the first nucleotide, then DNA
synthesis proceeds by a rather complex process to
ensure that the whole genome is copied.
In order to initiate the replication of many DNA
genomes, and some RNA genomes, a molecule of RNA
or protein is required to act as a primer.
7.3.1 RNA and protein primers
Synthesis of cell DNA commences after a region of the
double helix has been unwound by a helicase and
Cytoplasm Nucleus
Some Some
All
All
All
All
All
ssRNA dsDNA dsDNA ssRNA
→ →
 POLYMERASES 89

after a primase has synthesized short sequences of RNA Virus enzymes

complementary to regions of the DNA. These RNAs act
as primers; one is required for the leading strand, while
multiple primers must be synthesized for the Okazaki
fragments of the lagging strand. The first nucleotide of a
new sequence of DNA is linked to the 3 –OH group of
the primer RNA.
Some DNA viruses also use RNA primers during the
replication of their genomes. Some viruses, such as
polyomaviruses, use the cell primase to synthesize their
RNA primers, while others, such as herpesviruses and
phage T7, encode their own primases.
During their replication cycle the retroviruses
synthesize DNA from a (+) RNA template (Sec-tion
16.3.2). They use a cell transfer RNA to prime (−) DNA
synthesis, then they use the 3 –OH group in a
polypurine tract of the partly degraded (+) RNA
template to prime (+) DNA synthesis. The retrovirus
DNA becomes integrated into a cell chromosome. If the
infection is latent and the cell subsequently divides
(Section 9.3.1), then the virus DNA is copied along with
the cell DNA, using RNA primers synthesized by the
cell primase.
For some viruses the primer for initiation of nucleic
acid replication is the –OH group on a serine or tyrosine
residue in a protein. DNA viruses that use protein
primers include some animal viruses (e.g. adenoviruses)
and some phages (e.g. tectiviruses). RNA viruses that
use protein primers include some animal viruses (e.g.
picornaviruses) and some plant viruses (e.g.
luteoviruses).
Hepadnaviruses are DNA viruses that use a protein
primer to initiate (−) DNA synthesis and an RNA
primer to initiate (+) DNA synthesis (Section 18.8.6).
Protein primers (and the RNA primers of hepad-
naviruses) are not removed once their role is performed
and they are found linked to the 5 ends of the genomes
in virions (Section 3.2.3).
7.4 Polymerases
The key enzymes involved in virus genome replication
are DNA polymerases and RNA polymerases. Many
viruses encode their own polymerase, but some use a
host cell enzyme (Figure 7.3).
A DNA virus requires a DNA-dependent DNA
polymerase. Amongst the DNA viruses that replicate
DNA-dependent DNA polymerase
DNA DNA
RNA-dependent RNA polymerase
RNA RNA
RNA-dependent DNA polymerase
(reverse transcriptase)
RNA DNA
Cell enzymes
DNA-dependent DNA polymerase
DNA DNA
DNA-dependent RNA polymerase
(RNA pol II)
DNA RNA
Figure 7.3 Enzymes used by viruses to replicate their
genomes. Many viruses encode a polymerase to
replicate their genome, but some use a cell enzyme.
in the nuclei of eukaryotic cells, viruses with small
genomes (e.g. papillomaviruses) use the cell enzyme,
while viruses with large genomes (e.g. herpesviruses)
encode their own enzyme. Those DNA viruses that
replicate in the cytoplasm must encode their own
enzyme.
The enzyme that replicates the genome of an RNA
virus is often referred to as a replicase; for many RNA
viruses this is the same enzyme as that used for
transcription (Section 6.3.3). The retroviruses and the
pararetroviruses encode reverse transcriptases to
transcribe from RNA to DNA, and use the host cell
RNA polymerase II to transcribe from DNA to RNA.
Many viral polymerases form complexes with other
viral and/or cell proteins to produce the active enzyme.
Some of these additional proteins are processivity
factors, for example an Escherichia coli thioredoxin
molecule functions as a processivity factor for the DNA
polymerase of phage T7.
90 VIRUS GENOME REPLICATION

7.5 DNA replication
The viruses of Class I (dsDNA) and Class II (ssDNA)
replicate their genomes via dsDNA. The ssDNA viruses
first synthesize a complementary strand to convert the
genome into dsDNA.
Each viral DNA has at least one specific sequence
(ori ; replication origin) where replication is initiated.
The proteins that initiate DNA replication bind to this
site, and amongst these proteins are
• a helicase (unwinds the double helix at that site);
• a ssDNA binding protein (keeps the two strands
apart);
• a DNA polymerase.
Viral dsDNA is generally replicated by a process
similar to that used by cells to copy their genomes. The
basic process and the enzymes involved are outlined
in Figure 7.4. Fewer proteins are involved in bacterial
systems than in eukaryotic systems; for example, the
helicase – primase of phage T7 is a single protein
molecule, while that of herpes simplex virus is a
complex of three protein species.
DNA synthesis takes place near a replication fork.
One of the daughter strands is the leading strand and the
other is the lagging strand, synthesized as Okazaki
fragments, which become joined by a DNA ligase. After
a dsDNA molecule has been copied each of the daughter
molecules contains a strand of the original molecule.
This mode of replication is known as semi-conservative,
in contrast to the conservative replication of some
dsRNA viruses (Section 7.6).
Some DNA genomes are linear molecules, while
some are covalently closed circles (Section 3.2). Some
of the linear molecules are circularized prior to DNA
replication, hence many DNA genomes are replicated as
circular molecules, for which there are two modes of
replication, known as theta and sigma (Figure 7.5).
These terms refer to the shapes depicted in diagrams

5’
3’

DNA ligase lagging strand

DNA
polymerase Okazaki fragment
helicase-
primase
3’ 5’ RNA primer
5’
3’
DNA
replication
fork polymerase

leading strand

5’
Key: parental DNA 3’

new DNA
(arrow indicates synthesis
in 5' to 3' direction)

Figure 7.4 DNA replication. A helicase – primase unwinds the dsDNA and synthesizes RNA primers that are
used by the DNA polymerase to initiate DNA synthesis. The leading strand is synthesized continuously, while
the lagging strand is synthesized as Okazaki fragments that are joined together by a DNA ligase.
 DOUBLE-STRANDED RNA REPLICATION 91

ori

replication
fork
replication replication
fork fork

Key: ori origin of replication

parental DNA

new DNA
(arrow indicates synthesis
in 5' to 3' direction)

Figure 7.5 Theta and sigma modes of DNA replication. The theta structure is shown with two replication forks as
a result of bidirectional replication from ori; unidirectional replication can also give rise to a theta structure.

of the replicating molecules, which resemble the Greek
letters θ (theta) and σ (sigma). The sigma mode of
replication is also known as a rolling circle mode. The
genomes of some DNA viruses may be replicated by the
theta mode of replication early in infection and the
sigma mode late in infection.
Replication of the DNA of some viruses, such as
herpesviruses (Section 11.5.3) and phage T4, results in
the formation of very large DNA molecules called
concatemers. Each concatemer is composed of multiple
copies of the virus genome and the concatemers of
some viruses are branched. When DNA is packaged
during the assembly of a virion an endonuclease cuts a
genome length from a concatemer.
7.6 Double-stranded
RNA replication
Double-stranded RNA, like dsDNA, must be unwound
with a helicase in order for the molecule to be repli-
cated.
92 VIRUS GENOME REPLICATION
conservative
semi-conservative
Figure 7.6 Conservative and semi-conservative replication of dsRNA.
Some dsRNA viruses, e.g. Pseudomonas phage ϕ6 (ϕ
= Greek letter phi), replicate their genomes by a semi-
conservative mechanism, similar to dsDNA replication
(Section 7.5); each of the double-stranded progeny
molecules is made up of a parental strand and a
daughter strand. Other dsRNA viruses, includ-ing
members of the family Reoviridae (Chapter 13),
replicate by a mechanism designated as conservative
because the double-stranded molecule of the infecting
genome is conserved (Figure 7.6).
7.7 Single-stranded RNA
replication
The ssRNA genomes of viruses in Classes IV and V are
replicated by synthesis of complementary strands of
RNA that are then used as templates for synthesis of
new copies of the genome (Figure 7.1). The synthesis of
each RNA molecule requires the recruitment of an
RNA-dependent RNA polymerase to the 3 end of the
template, therefore both plus- and minus-strand RNA
must have a binding site for the enzyme at the 3 end.
An interesting point to note here is that all class IV
viruses of eukaryotes replicate their RNA in asso-ciation
with cytoplasmic membranes. For many groups of
viruses, including picornaviruses (Section 14.4.4), these
membranes are derived mainly from the endo-plasmic
reticulum, but other membranous structures are
Key: (+) RNA
(–) RNA parental strands
(+) RNA
(–) RNA daughter strands
used, including endosomes (by togaviruses) and chloro-
plasts (by tombusviruses). Viral proteins, including the
RNA polymerases, are bound to the membranes.
During the replication of ssRNA both (+) and (−)
strands of RNA accumulate in the infected cell, but not
in equal amounts. Plus-strand RNA viruses accumulate
an excess of (+) RNA over (−) RNA, and for minus-
strand RNA viruses the reverse is true.
7.8 Reverse transcription
Some RNA viruses replicate their genomes via a DNA
intermediate, while some DNA viruses replicate their
genomes via an RNA intermediate (Figure 7.1). Both of
these modes of genome replication involve reverse
transcription, which has two major steps: synthesis of
(−) DNA from a (+) RNA template followed by
synthesis of a second DNA strand (Figure 7.7). Both
steps are catalysed by a reverse transcriptase that is
encoded by the virus.
Reverse transcription takes place within a viral
structure in the cytoplasm of the infected cell. In later
chapters the process is considered in more detail for the
retroviruses (Section 16.3.2) and for hepatitis B virus
(Section 18.8.6). No viruses of prokaryotes are known
to carry out reverse transcription.
 SOURCES OF FURTHER INFORMATION 93

(+) RNA
RT
(–) DNA
RT Sources of further information
+ dsDNA
–
Books
Figure 7.7 Reverse transcription. ssDNA is Brown T. A. (2002) Chapter 13 in Genomes, 2nd edition,
synthe-sized from an RNA template then the BIOS
ssDNA is con-verted to dsDNA. A reverse Cann A. J., editor (2000) DNA Virus Replication, Oxford
transcriptase (RT) carries out both steps. University Press
Learning outcomes
By the end of this chapter you should be able to

• state the locations within eukaryotic cells where different categories of virus genome are repli-cated;

• explain the role of primers in virus nucleic acid synthesis;

• discuss the roles of virus and host proteins in virus genome replication;

• outline the replication mechanisms of virus DNAs and RNAs;

• explain the term ‘reverse transcription’.

Journal
van Dijk A. A. et al. (2004) Initiation of viral RNA-dependent RNA polymerization Journal of General Virol-ogy, 85, 1077 –
1093