Rolling hairpin replication


Rolling hairpin replication is a unidirectional, strand displacement form of DNA replication used by parvoviruses, a group of viruses that constitute the family Parvoviridae. Parvoviruses have linear, single-stranded DNA genomes in which the coding portion of the genome is flanked by telomeres at both ends that form hairpin loops. During RHR, these hairpins unfold and refold to change the direction of DNA replication to progress in a continuous manner back and forth across the genome. This creates a replicative form molecule that contains numerous copies of the genome. Progeny genomes are then excised from the RF molecule by excisions made by the viral replication initiator protein. RHR is similar to rolling circle replication and can be considered a variation of it used for linear ssDNA genomes.
Before RHR begins, a host cell DNA polymerase converts the genome to a duplex form in which the coding portion is double-stranded and connected to the terminal hairpins. Messenger RNA that encodes the viral initiator protein is then transcribed and translated to synthesize the protein. The initiator protein commences RHR by binding to and nicking the genome in a region at the base of the hairpin called the origin while establishing a replication fork. Nicking leads to the hairpin unfolding into a linear, extended form. The telomere is then replicated and both strands of the telomere refold back in on themselves to their original hairpin forms, which repositions the replication fork to switch templates to the other strand and move in the opposite direction to the other end of the RF molecule.
Parvoviruses vary in whether both hairpins are the same or different. Homotelomeric parvoviruses such as adeno-associated viruses, i.e. those that have similar or identical telomeres, have both ends replicated by terminal resolution, the previously described process. Heterotelomeric parvoviruses such as minute virus of mice, i.e. those that have different telomeres, have one end replicated by terminal resolution and the other end by an asymmetric process called junction resolution. During asymmetric junction resolution, the duplex extended form of the telomere reorganizes into a cruciform-shaped junction, which enables the correct orientation of the telomere to be replicated off the lower arm of the cruciform. Because of their means of resolving termini, homotelomeric parvoviruses usually package an equal number of positive- and negative-sense strands into progeny capsids, while heterotelomeric parvoviruses typically package negative-sense strands.

Introduction

Parvoviruses

Parvoviruses are a family of DNA viruses, family Parvoviridae, that have single-stranded DNA genomes enclosed in rugged, icosahedral capsids that are 18–26 nanometers in diameter. They have linear genomes with short terminal sequences at each end of the genome. These termini are imperfect palindromes of nucleotides able to fold into structures called hairpins, or hairpin loops. Varying from virus to virus, the coding region of the genome is 4–6 kilobases in length, and the termini are 116–550 nucleotides in length each. The telomere sequences give rise to origins of replication and contain most of the cis-acting information needed for DNA replication and packaging. Parvoviruses have two genes: rep on the "left" half, which encodes non-structural proteins involved in replication and transcription, and cap on the "right" half, which encodes capsid proteins.
Parvovirus genomes may be either positive-sense or negative-sense. Some species, such as adeno-associated viruses like AAV2, package a roughly equal number of positive-sense and negative-sense strands into virions. Others, such as minute virus of mice, mainly package negative-sense strands, while others have varying proportions. To clarify this disparity, the 5′ end of the strand that encodes the non-structural proteins is called the left end, and the 3′ end is called the right end. For the negative-sense strand, the 3′ end is the left end and the 5′ end is the right end. AAV and MVM have been the main viruses used to study parvovirus replication.

Rolling hairpin replication

Parvoviruses replicate their genomes through a process called rolling hairpin replication, which is a unidirectional, strand displacement form of DNA replication. Before replication, the coding portion of the ssDNA genome is converted to a double-strand DNA form, which is then cleaved by a viral protein to initiate replication. Sequential unfolding and refolding of the hairpins acts to reverse the direction of synthesis, which allows replication to go back and forth along the genome to synthesize a continuous duplex replicative form DNA intermediate. The viral replication initiator protein then excises progeny ssDNA genomes from the RF intermediate. Between genera, parvoviral hairpins vary significantly, but they are well conserved within genera.
Parvovirus genomes have distinct starting points of replication that contain palindromic DNA sequences. These sequences are able to alternate between inter- and intrastrand basepairing throughout replication, and they serve as self-priming telomeres at each end of the DNA molecule. They also contain two key sites necessary for replication that are used by the initiator protein: a binding site and a cleavage site. Telomere sequences have significant complexity and diversity, suggesting that they perform additional functions for many species. In general, however, they have the same basic structure: imperfect palindromes in which a fully or primarily basepaired region terminates into axial symmetry. These termini can fold into a variety of structures such as a Y-shaped structure and a cruciform-shaped structure. During replication, they act as hinges that unfold and refold the hairpin, enabled by the imperfectly basepaired or partial cruciform regions surrounding the axis. Hairpin unfolding and refolding require the viral replication initiator protein, which binds site-specifically to duplex motifs in telomeres and contains a superfamily 3 helicase domain to help melt dsDNA.
Some parvoviruses, such as AAV2, are homotelomeric, which means the two palindromic telomeres are similar or identical. Replication at each terminal ending is therefore symmetrical. Other parvoviruses, such as MVM, are heterotelomeric, which means they have two physically different telomeres. As a result, heterotelomeric parvoviruses have a more complex replication cycle since the two telomeres are replicated through different processes. Homotelomeric parvoviruses replicate both ends through a process called terminal resolution, whereas heterotelomeric parvoviruses replicate one end by terminal resolution and the other end by an asymmetric process called junction resolution. Replication occurs in two phases: the first amplifies the number of copies of the genome by creating long palindromic concatemers, and the second involves excision and displacement of individual progeny ssDNA genomes from the concatemer for further replication or packaging into progeny capsids.

Summary

The general process of rolling hairpin replication can be summarized as follows:
  • 1. The coding portion of the genome is replicated by a host DNA polymerase, starting from the 3′ end of the 3′ hairpin, which acts as a primer. Replication continues until the newly synthesized strand is connected to the 5′ end of the 5′ hairpin, which produces a duplex DNA molecule that has two strands of the coding portion of the genome, called a replicative form molecule.
  • 2. mRNA that encodes the viral replication initiator protein is transcribed and subsequently translated to synthesize the protein.
  • 3. The initiator protein binds to and, with its endonuclease activity, cleaves the DNA within a region called the origin, which results in a hairpin unfolding into a linear, extended form. At the same time, the initiator protein establishes a replication fork with its helicase activity.
  • 4. The extended-form hairpin is replicated to create an inverted copy of the unfolded hairpin on the newly synthesized strand.
  • 5. The two strands of that end refold back into two hairpins, which repositions the replication fork to switch templates and move in the opposite direction.
  • 6. DNA replication continues in a linear manner from one end of the replicative molecule to the other.
  • 7. Upon reaching the other end, that end's hairpin is unfolded and refolded to replicate the hairpin and once again swap templates and change the direction of replication. This back-and-forth replication is continually repeated, which produces a concatemer of numerous copies of the genome.
  • 8. The viral initiator protein excises individual genomic strands of DNA from the replicative concatemer by nicking origins, which attaches the initiator protein to the 5′ end of the nicked strand and frees a 3′ hydroxyl, which can prime replication, while displacing a progeny ssDNA strand.
  • 9. Excised ssDNA genomes are packaged into newly constructed viral capsids, or their telomeres may fold into hairpins as they are recycled for further replication.
  • 10. RF molecules are able to support multiple replication forks, which may be established at nicked origins. For homotelomeric parvoviruses, both telomeres are replicated by terminal resolution after nicking, while for heterotelomeric parvoviruses, one end is replicated through terminal resolution after nicking and one end by junction resolution after nicking.

    Preparation for replication

The viral replication initiator protein is attached on the outside of the virion to the 5′ end of the viral genome on the inside by a tether about 24 nucleotides in length. During cell entry, this tether is cleaved off to be restored later, which releases the initiator protein from the genomic molecule. After cell entry, virions accumulate in the cell nucleus with the genome still contained in the capsid. These capsids may be reconfigured to an open, or "transitioned", state during entry. How and when the genome leaves the capsid is unclear. For AAV, it has been suggested that nuclear factors disassemble the capsid, but analysis of MVM implies that the genome is ejected in a 3′-to-5′ direction from an opening in the capsid called a portal.
Naked ssDNA is likely to be unstable, perceived as foreign by the host cell, or improperly replicated by host DNA repair. For that reason, the genome must either be converted rapidly to its less obstructive, more stable duplex form or retained within the capsid until it is uncoated during the host cell's DNA synthesis phase. Parvoviruses were traditionally thought to wait for S-phase, but more recent evidence suggests that parvoviral nonstructural proteins may influence the host cell cycle. Until S-phase begins, virions are retained in the nucleus and may make use of certain strategies to evade host defense mechanisms to protect their genomes until S-phase, though it is unclear how this occurs. Since the genome is packaged as ssDNA, creation of a complementary strand to create a double-stranded DNA molecule is necessary before gene expression.
DNA polymerases are only able to synthesize DNA in a 5′ to 3′ direction, and they require a basepaired primer to begin synthesis. Parvoviruses address these limitations by using their termini as primers for complementary strand synthesis. For MVM, this is the left-end telomere, which folds back on itself to create a 3′-OH end of the left-hand terminus that pairs with an internal base. This primes initial DNA synthesis for a host DNA polymerase to convert the ssDNA genome to its first dsDNA form. This is a duplex molecule in which both strands are covalently cross-linked to each other at the left end by a copy of the telomere. Synthesis of this duplex form precedes expression of the initiator protein so that when the replication fork during initial complementary strand synthesis reaches the right-hand end, it does not displace and copy the right-end hairpin. This allows the new DNA strand's 3′ end to be ligated to the right hairpin's 5′ end by a host ligase, thereby creating a covalently continuous duplex molecule. Furthermore, cellular machinery is unable to melt and copy the 5′ hairpin, so expression of the initiator protein must precede replication. During this step, the tether sequence that was present before viral entry into the cell is resynthesized.