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Virus genome replication

At a glance

DdDp/DdDp 1


RdDp DdRp

DdRp RdDp

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)

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)

however. while Class some RNA viruses that replicate their genomes via a VI viruses replicate via a DNA intermediate.1 Overview of virus DNA intermediate. DNA viruses copy their genomes directly converted to dsDNA prior to copying.1). Plus strand genomes have the same sequence as genome of the infecting virus is replicated so that viral the mRNA (except that in DNA thymine replaces transcription can be amplified and to provide copies of uracil). to DNA and RNA viruses copy their genomes directly There are two classes of viruses with (+) RNA to RNA. The various replication modes of virus genomes are summarized in Figure 7.1. some DNA viruses that genomes (Figure 7. Single-stranded DNA is Generally.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. complementary to the mRNA. The . Class IV viruses copy their (+) replicate their genomes via an RNA intermediate and RNA genomes via a (−) RNA intermediate. genome replication Single-stranded genomes are designated as plus or In this chapter we consider the fifth step of our minus depending on their relationship to the virus generalized replication cycle: genome replication. There are. while minus-strand genomes have the sequence the genome for progeny virions. The mRNA.

Most of these viruses have either a (+) or a (−) strand genome. . (+) RNA and (+) DNA have the same sequence as the mRNA (except that in DNA thymine replaces uracil). 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. Some ssDNA viruses and some ssRNA viruses have ambisense genomes. (−) RNA and (−) DNA have the sequence complementary to the mRNA (except that in DNA thymine replaces uracil). (+) and (−) strands are indicated for the ssDNA of the Class II viruses. (+) 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.1 Replication of virus genomes in the seven Baltimore Classes.

Some ssDNA viruses. and then we shall give individual attention to replication of the genomes of the Figure 7. then DNA minus-strand RNA viruses with segmented genomes are synthesis proceeds by a rather complex process to replicated in the nucleus. As we saw in Chapter 5. varies with the type of genome linked to the second nucleotide. use self- The genomes of most DNA viruses are replicated in priming.1 RNA and protein primers Each virus genome has a specific sequence where nucleic acid replication is initiated. such as parvoviruses.88 VIRUS GENOME REPLICATION synthesis of DNA from an RNA template (reverse tran. but those of some dsDNA viruses are complementary sequences that can base pair (Figure replicated in the cytoplasm. 7. (Table 7.1 Locations of virus genome replication in eukaryotic cells Virus genome Cytoplasm Nucleus dsDNA Some Some ssDNA All dsRNA All (+) RNA All (−) RNA (non-segmented genome) All (−) RNA (segmented genome) All Retroviruses [(+) RNA] ssRNA dsDNA dsDNA ssRNA Pararetroviruses [dsDNA] → → . a molecule of RNA nucleus. Nucleic acid replication requires priming. when viruses infect eukaryotic and rhabdoviruses) initiates when the first nucleotide of cells the genomes of some are delivered to the the new strand base pairs with a nucleotide in the viral cytoplasm and some are conveyed to the nucleus.1).2 3 end of a ssDNA where self-priming DNA viruses. The retroviruses and ensure that the whole genome is copied. At the 3 end of the DNA there are regions with the nucleus. transcribing viruses. The initial nucleotide effectively acts as a primer destination of a virus genome. OH In this chapter we shall look at some general aspects of virus genome replication. Replication of the replication in eukaryotic cells genomes of many RNA viruses (including rotaviruses.2). 3’ scription) is also a characteristic of Class VII viruses. pararetroviruses are special cases: each replicates RNA In order to initiate the replication of many DNA to DNA in the cytoplasm and DNA to RNA in the genomes. The –OH group of the nucleotide at the 3 end viruses are replicated in the cytoplasm. but those of the forms a linkage with the first nucleotide. the RNA viruses and the reverse of DNA synthesis can occur. The RNA.3. The genomes of most RNA 7.3 Initiation of genome replication 7. and some RNA genomes. or protein is required to act as a primer. double helix has been unwound by a helicase and Table 7. and hence the location in for RNA replication when its 3 –OH group becomes which it is replicated.2 Locations of virus genome the first reaction of a nucleotide with an –OH group on a molecule at the initiation site. This sequence is Synthesis of cell DNA commences after a region of the recognized by the proteins that initiate replication. which is 7.

for example an Escherichia coli thioredoxin A DNA virus requires a DNA-dependent DNA molecule functions as a processivity factor for the DNA polymerase. factors. tectiviruses).3. They use a cell transfer RNA to prime (−) DNA synthesis. Some viruses. use the cell primase to synthesize their RNA-dependent DNA polymerase RNA primers. but some use a cell enzyme.3.3). The retroviruses and the pararetroviruses encode reverse transcriptases to 7. These RNAs act as primers. enzyme. RNA viruses that use protein primers include some animal viruses (e.2. adenoviruses) and some phages (e. The retrovirus DNA DNA DNA becomes integrated into a cell chromosome.3.g. one is required for the leading strand. viruses encode their own polymerase. using RNA primers synthesized by the DNA RNA cell primase. POLYMERASES 89 after a primase has synthesized short sequences of RNA Virus enzymes complementary to regions of the DNA. then they use the 3 –OH group in a Cell enzymes polypurine tract of the partly degraded (+) RNA DNA-dependent DNA polymerase template to prime (+) DNA synthesis. Amongst the DNA viruses that replicate polymerase of phage T7. papillomaviruses) use the cell enzyme. then the virus DNA is copied along with DNA-dependent RNA polymerase (RNA pol II) the cell DNA. while viruses with large genomes (e. For some viruses the primer for initiation of nucleic Figure 7.g. such as herpesviruses and (reverse transcriptase) phage T7. The enzyme that replicates the genome of an RNA naviruses) are not removed once their role is performed virus is often referred to as a replicase. such as polyomaviruses.2).3). herpesviruses) Hepadnaviruses are DNA viruses that use a protein encode their own enzyme. but some use a Some of these additional proteins are processivity host cell enzyme (Figure 7. If the infection is latent and the cell subsequently divides (Section 9.1). DNA viruses that use protein replicate their genome. RNA RNA Some DNA viruses also use RNA primers during the replication of their genomes. Many viral and/or cell proteins to produce the active enzyme. Those DNA viruses that primer to initiate (−) DNA synthesis and an RNA replicate in the cytoplasm must encode their own primer to initiate (+) DNA synthesis (Section 18.3).6). and use the host cell RNA polymerase II to transcribe from DNA to RNA.g. transcription (Section 6. The key enzymes involved in virus genome replication Many viral polymerases form complexes with other are DNA polymerases and RNA polymerases.4 Polymerases transcribe from RNA to DNA. viruses with small picornaviruses) and some plant viruses (e. encode their own primases. .3 Enzymes used by viruses to replicate their acid replication is the –OH group on a serine or tyrosine genomes. RNA DNA During their replication cycle the retroviruses synthesize DNA from a (+) RNA template (Sec-tion 16. primers include some animal viruses (e.g. for many RNA and they are found linked to the 5 ends of the genomes viruses this is the same enzyme as that used for in virions (Section 3. Protein primers (and the RNA primers of hepad. Many viruses encode a polymerase to residue in a protein. while DNA-dependent DNA polymerase DNA DNA multiple primers must be synthesized for the Okazaki fragments of the lagging strand.g.8. while others. in the nuclei of eukaryotic cells. genomes (e. The first nucleotide of a new sequence of DNA is linked to the 3 –OH group of RNA-dependent RNA polymerase the primer RNA.g. luteoviruses).

for which there are two modes of similar to that used by cells to copy their genomes.2). The replication. Some DNA genomes are linear molecules. Fewer proteins are involved in bacterial systems than in eukaryotic systems. Each viral DNA has at least one specific sequence One of the daughter strands is the leading strand and the (ori . This mode of replication is known as semi-conservative. DNA synthesis takes place near a replication fork. other is the lagging strand. the The viruses of Class I (dsDNA) and Class II (ssDNA) helicase – primase of phage T7 is a single protein replicate their genomes via dsDNA. of the linear molecules are circularized prior to DNA replication.6). for example. while some are covalently closed circles (Section 3.4 DNA replication. while that of herpes simplex virus is a first synthesize a complementary strand to convert the complex of three protein species. • a helicase (unwinds the double helix at that site). genome into dsDNA. known as theta and sigma (Figure 7. The leading strand is synthesized continuously.90 VIRUS GENOME REPLICATION 7. Some • a DNA polymerase. After site.5). and amongst these proteins are a dsDNA molecule has been copied each of the daughter molecules contains a strand of the original molecule. in contrast to the conservative replication of some • a ssDNA binding protein (keeps the two strands dsRNA viruses (Section 7. synthesized as Okazaki The proteins that initiate DNA replication bind to this fragments. A helicase – primase unwinds the dsDNA and synthesizes RNA primers that are used by the DNA polymerase to initiate DNA synthesis. replication origin) where replication is initiated. hence many DNA genomes are replicated as Viral dsDNA is generally replicated by a process circular molecules. while the lagging strand is synthesized as Okazaki fragments that are joined together by a DNA ligase. . which become joined by a DNA ligase. apart). The ssDNA viruses molecule. basic process and the enzymes involved are outlined 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 7.4.5 DNA replication in Figure 7.

6 Double-stranded Replication of the DNA of some viruses.3) and phage T4. of the replicating molecules.5. The genome length from a concatemer. The sigma mode of during the assembly of a virion an endonuclease cuts a replication is also known as a rolling circle mode. like dsDNA. such as RNA replication herpesviruses (Section 11. 7. . Each concatemer is composed of multiple with a helicase in order for the molecule to be repli- copies of the virus genome and the concatemers of cated. The theta structure is shown with two replication forks as a result of bidirectional replication from ori. results in the formation of very large DNA molecules called Double-stranded RNA. unidirectional replication can also give rise to a theta structure.5 Theta and sigma modes of DNA replication. which resemble the Greek some viruses are branched. genomes of some DNA viruses may be replicated by the theta mode of replication early in infection and the sigma mode late in infection. must be unwound concatemers. When DNA is packaged letters θ (theta) and σ (sigma). 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.

(Section 7. No viruses of prokaryotes are known membranes are derived mainly from the endo-plasmic to carry out reverse transcription. must have a binding site for the enzyme at the 3 end. because the double-stranded molecule of the infecting genome is conserved (Figure 7. Plus-strand RNA viruses accumulate members of the family Reoviridae (Chapter 13).g. Some dsRNA viruses. The synthesis of (−) DNA from a (+) RNA template followed by each RNA molecule requires the recruitment of an synthesis of a second DNA strand (Figure 7. similar to dsDNA replication RNA polymerases. For many groups of retroviruses (Section 16.2) and for hepatitis B virus viruses.1).3. while some DNA viruses replicate their The ssRNA genomes of viruses in Classes IV and V are genomes via an RNA intermediate (Figure 7. Pseudomonas phage ϕ6 (ϕ used. includ-ing in equal amounts. these (Section 18. are bound to the membranes. which has two major steps: synthesis of new copies of the genome (Figure 7. Other dsRNA viruses.6).7 Single-stranded RNA Some RNA viruses replicate their genomes via a DNA replication intermediate. plasts (by tombusviruses). including picornaviruses (Section 14.92 VIRUS GENOME REPLICATION conservative semi-conservative Key: (+) RNA (–) RNA parental strands (+) RNA (–) RNA daughter strands Figure 7.and minus-strand RNA encoded by the virus. reticulum.4). Both RNA-dependent RNA polymerase to the 3 end of the steps are catalysed by a reverse transcriptase that is template. each of the double-stranded progeny During the replication of ssRNA both (+) and (−) molecules is made up of a parental strand and a strands of RNA accumulate in the infected cell. Reverse transcription takes place within a viral An interesting point to note here is that all class IV structure in the cytoplasm of the infected cell. In later viruses of eukaryotes replicate their RNA in asso-ciation chapters the process is considered in more detail for the with cytoplasmic membranes.8 Reverse transcription 7. Both of replicated by synthesis of complementary strands of these modes of genome replication involve reverse RNA that are then used as templates for synthesis of transcription.4. but not daughter strand. but other membranous structures are . replicate their genomes by a semi. 7.5).1).8. an excess of (+) RNA over (−) RNA. including the conservative mechanism.7).6). and for minus- replicate by a mechanism designated as conservative strand RNA viruses the reverse is true.6 Conservative and semi-conservative replication of dsRNA. including endosomes (by togaviruses) and chloro- = Greek letter phi). e. Viral proteins. therefore both plus.

synthe-sized from an RNA template then the BIOS ssDNA is con-verted to dsDNA. A. A reverse Cann A. Oxford transcriptase (RT) carries out both steps. J. SOURCES OF FURTHER INFORMATION 93 (+) RNA RT (–) DNA RT Sources of further information + dsDNA – Books Figure 7. 2nd edition. (2002) Chapter 13 in Genomes.7 Reverse transcription. editor (2000) DNA Virus Replication. University Press .. ssDNA is Brown T.

.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 term ‘reverse transcription’. • discuss the roles of virus and host proteins in virus genome replication. • outline the replication mechanisms of virus DNAs and RNAs. • explain the role of primers in virus nucleic acid synthesis.

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