Virus Genome Replication PDF

6/20/2017 John B. Carter and Venetia A. Saunders Figures Chapter 7 © 2012 John Wiley & Sons Ltd. 1...

6/20/2017 John B. Carter and Venetia A. Saunders Figures Chapter 7 © 2012 John Wiley & Sons Ltd. 1 www.wiley.com/college/carter 7.1 Overview of virus 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 DNA intermediate. The various replication modes of virus genomes are summarized in Figure 7.1. © 2012 John Wiley & Sons Ltd. 2 www.wiley.com/college/carter 1  6/20/2017 © 2012 John Wiley & Sons Ltd. 3 www.wiley.com/college/carter Single-stranded genomes are designated as plus or minus depending on their relationship to the virus mRNA. + strand genomes have the same sequence as the mRNA (except that in DNA thymine replaces uracil), while - 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. Class VI viruses replicate via a DNA intermediate. © 2012 John Wiley & Sons Ltd. 4 www.wiley.com/college/carter 2  6/20/2017 The synthesis of DNA from an RNA template (reverse transcription) 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. © 2012 John Wiley & Sons Ltd. 5 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 6 www.wiley.com/college/carter 3  6/20/2017 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 All genome) (−) RNA (segmented All genome) Retroviruses [(+) RNA] ssRNA → dsDNA dsDNA → ssRNA Pararetroviruses [dsDNA] ssRNA → dsDNA dsDNA → ssRNA © 2012 John Wiley & Sons Ltd. 7 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 8 www.wiley.com/college/carter 4  6/20/2017 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. 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. © 2012 John Wiley & Sons Ltd. 9 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 10 www.wiley.com/college/carter 5  6/20/2017 © 2012 John Wiley & Sons Ltd. 11 www.wiley.com/college/carter 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 after a primase has synthesized short sequences of RNA 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. © 2012 John Wiley & Sons Ltd. 12 www.wiley.com/college/carter 6  6/20/2017 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. Others, such as herpesviruses and phage T7, encode their own primases. During their replication cycle the retroviruses synthesize DNA from a (+) RNA template (Section 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. © 2012 John Wiley & Sons Ltd. 13 www.wiley.com/college/carter 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). © 2012 John Wiley & Sons Ltd. 14 www.wiley.com/college/carter 7  6/20/2017 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 hepadnaviruses) 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). © 2012 John Wiley & Sons Ltd. 15 www.wiley.com/college/carter 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 7.4). A DNA virus requires a DNA-dependent DNA polymerase. Amongst the DNA viruses that replicate 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. © 2012 John Wiley & Sons Ltd. 16 www.wiley.com/college/carter 8  6/20/2017 © 2012 John Wiley & Sons Ltd. 17 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 18 www.wiley.com/college/carter 9  6/20/2017 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. © 2012 John Wiley & Sons Ltd. 19 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 20 www.wiley.com/college/carter 10  6/20/2017 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 7.3. 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. © 2012 John Wiley & Sons Ltd. 21 www.wiley.com/college/carter © 2012 John Wiley & Sons Ltd. 22 www.wiley.com/college/carter 11  6/20/2017 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 semiconservative, in contrast to the conservative replication of some dsRNA viruses (Section 7.6). © 2012 John Wiley & Sons Ltd. 23 www.wiley.com/college/carter 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 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. © 2012 John Wiley & Sons Ltd. 24 www.wiley.com/college/carter 12  6/20/2017 © 2012 John Wiley & Sons Ltd. 25 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 26 www.wiley.com/college/carter 13  6/20/2017 7.6 Double-stranded RNA replication Double-stranded RNA, like dsDNA, must be unwound with a helicase in order for the molecule to be replicated. 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, including 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). © 2012 John Wiley & Sons Ltd. 27 www.wiley.com/college/carter © 2012 John Wiley & Sons Ltd. 28 www.wiley.com/college/carter 14  6/20/2017 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. © 2012 John Wiley & Sons Ltd. 29 www.wiley.com/college/carter © 2012 John Wiley & Sons Ltd. 30 www.wiley.com/college/carter 15  6/20/2017 An interesting point to note here is that all class IV viruses of eukaryotes replicate their RNA in association with cytoplasmic membranes. For many groups of viruses, including picornaviruses (Section 14.4.4), these membranes are derived mainly from the endoplasmic reticulum. But other membranous structures are used, including endosomes (by togaviruses) and chloroplasts (by tombusviruses). Viral proteins, including the RNA polymerases, are bound to the membranes. © 2012 John Wiley & Sons Ltd. 31 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 32 www.wiley.com/college/carter 16  6/20/2017 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. © 2012 John Wiley & Sons Ltd. 33 www.wiley.com/college/carter © 2012 John Wiley & Sons Ltd. 34 www.wiley.com/college/carter 17  6/20/2017 © 2012 John Wiley & Sons Ltd. 35 www.wiley.com/college/carter 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. © 2012 John Wiley & Sons Ltd. 36 www.wiley.com/college/carter 18  6/20/2017 Thank You… © 2012 John Wiley & Sons Ltd. 37 www.wiley.com/college/carter 19