Rolling hairpin replication (RHR) 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 (ssDNA) genomes in which the coding portion of the genome is flanked by telomeres at each end that form hairpin loops. During RHR, these hairpin loops repeatedly unfold and refold to change the direction of DNA replication so that replication progresses in a continuous manner back and forth across the genome. RHR is initiated and terminated by an endonuclease encoded by parvoviruses that is variously called NS1 or Rep, and RHR is similar to rolling circle replication, which is used by ssDNA viruses that have circular 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. From there, messenger RNA (mRNA) that encodes the viral initiator protein is transcribed and translated to synthesize the protein. The initiator protein commences RHR by binding to and nicking the genome in a region adjacent to a hairpin called the origin and establishing a replication fork with its helicase activity. 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 turn-around forms. This repositions the replication fork to switch templates to the other strand and move in the opposite direction. Upon reaching the other end, the same process of unfolding, replication, and refolding occurs.
Parvoviruses vary in whether both hairpins are the same or different. Homotelomeric parvoviruses such as adeno-associated viruses (AAV), i.e. those that have identical or similar telomeres, have both ends replicated by terminal resolution, the previously described process. Heterotelomeric parvoviruses such as minute virus of mice (MVM), i.e. those that have different telomeres, have one end replicated by terminal resolution and the other by an asymmetric process called junction resolution. During asymmetric junction resolution, the duplex extended form of the telomere reorganizes into a cruciform-shaped junction, and the correct orientation of the telomere is replicated off the lower arm of the cruciform. As a result of RHR, a replicative molecule that contains numerous copies of the genomes is synthesized. The initiator protein periodically excises progeny ssDNA genomes from this replicative concatemer.
Background.
Parvoviruses are a family of DNA viruses that have single-stranded DNA (ssDNA) genomes enclosed in rugged, icosahedral protein capsids 18–26 nanometers (nm) in diameter. Unlike most other ssDNA viruses, which have circular genomes that form a loop, parvoviruses have linear genomes with short terminal sequences at each end of the genome. These termini are capable of being formed into structures called hairpins or hairpin loops and consist of short, imperfect palindromes. Varying from virus to virus, the coding region of the genome is 4–6 kilobases (kb) in length, and the termini are 116–550 nucleotides (nt) in length each. The hairpin sequences provide most of the "cis"-acting information needed for DNA replication and packaging.
Parvovirus genomes may be either positive-sense or negative-sense. Some species, such as adeno-associated viruses (AAV) like AAV2, package a roughly equal number of positive-sense and negative-sense strands into virions, others, such as minute virus of mice (MVM), show preference toward packaging negative-sense strands, and others have varying proportions. Because of this disparity, the 5′-end (usually pronounced "five prime end") of the strand that encodes the non-structural proteins is called the "left end", and the 3′-end (usually pronounced "three prime end") is called the "right end". In reference to the negative-sense strand, the 3′-end is the left side and the 5′-end is the right side.
Parvoviruses replicate their genomes through a process called rolling hairpin replication (RHR), 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 (dsDNA) form, which is then cleaved by a viral protein to initiate replication. Sequential unfolding and refolding of the hairpin termini 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 (RF) DNA intermediate. Progeny ssDNA genomes are then excised from the RF intermediate. While the general aspects of RHR are conserved across genera and species, the exact details likely vary.
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 genome. They also contain two key sites necessary for replication 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 MVM, for example, the left-end hairpin contains binding sites for transcription factors that modulate gene expression from an adjacent promoter. For AAV, the hairpins can bind to MRE11/Rad50/NBS1 (MRN) complexes and Ku70/80 heterodimers, which are involved in sensing and repairing DNA. In general, however, they have the same basic structure: imperfect palindromes in which a fully or primarily basepaired region terminates into an axial symmetry. These palindromes can fold into a variety of structures such as a Y-shaped structure and a cruciform-shaped structure. During replication, the termini act as hinges in which the imperfectly basepaired or partial cruciform regions surrounding the axis provide a favorable environment for unfolding and refolding of the hairpin.
Some parvoviruses, such as AAV2, are homotelomeric, meaning the two palindromic telomeres are similar or identical and form part of larger (inverted) terminal repeat ((I)TR) sequences. Replication at each terminal ending is therefore similar. Other parvoviruses, such as MVM, are heterotelomeric, meaning they have two physically different telomeres. As a result, heterotelomeric parvoviruses tend to have a more complex replication process since the two telomeres have different replication processes. In general, homotelomeric parvoviruses replicate both ends via 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. Whether a genus is hetero- or homotelomeric, along with other genomic characteristics, is shown in the following table.
General process.
The entire process of rolling hairpin replication, which has distinct, sequential stages, can be summarized as follows:
Preparation for replication.
Upon cell entry, a tether about 24 nucleotides in length that attaches the viral protein NS1, essential in replication, to the virion is cleaved off the virion to be reattached later. After cell entry, virions accumulate in the cell nucleus while the genome is still contained within the capsid. These capsids may be reconfigured to an open or transitioned state during entry. The exact mechanism by which the genome leaves the capsid is unclear. For AAV, it has been suggested that nuclear factors disassemble the capsid, whereas for MVM, it appears as if the genome is ejected in a 3′-to-5′ direction from an opening in the capsid called a portal.
Parvoviruses lack genes capable of inducing resting cells to enter their DNA synthesis phase (S-phase). Additionally, naked ssDNA is likely to be unstable, perceived as foreign by the host cell, or improperly replicated by host DNA repair. For these reasons, 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 S-phase. Typically, the latter occurs and virion remains silent in the nucleus until the host cell enters S-phase by itself. During this waiting period, virions may make use of certain strategies to evade host defense mechanisms to protect their hairpins and DNA to reach S-phase, though it is unclear how this occurs. Since the genome is packaged as ssDNA, creation of a complementary strand is necessary before gene expression.
DNA polymerases are only able to synthesize DNA in a 5′ to 3′ direction, and they require a basepair primer to begin synthesis. Parvoviruses address these limitations by using their termini as primers for complementary strand synthesis. A 3′ hydroxyl end of the left-hand (3′) terminus pairs with an internal base to prime initial DNA synthesis, resulting in the conversion of the ssDNA genome to its first duplex form. This is a monomeric double-stranded DNA molecule in which the two strands are covalently cross-linked to each other at the left-end by a single copy of the viral telomere. Synthesis of the duplex form precedes NS1 expression so that when the replication fork during initial complementary strand synthesis reaches the right (5′) end, it does not displace and copy the right-end hairpin. This allows the 3′-end of the new DNA strand to be covalently ligated to the 5′-end of the right hairpin by a host ligase, thereby creating the duplex molecule. During this step, the tether sequence that was present before viral entry into the cell is resynthesized.
Essential viral proteins and initiation.
Once an infected cell enters S-phase, parvovirus genomes are converted to their duplex form by host replication machinery, and mRNA that encodes non-structural (NS) proteins is transcribed starting from a viral promoter (P4 for MVM). One of these NS proteins is usually called NS1 but also Rep1 or Rep68/78 for the genus "Dependoparvovirus", which AAV belongs to. NS1 is a site-specific DNA binding protein that acts as the replication initiator protein via nickase activity. It also mediates excision of both ends of the genome from duplex RF intermediates via a transesterification reaction that introduces a nick into specific duplex origin sequences. Key components of NS1 include an HUH endonuclease domain toward the N-terminus of the protein and a superfamily 3 (SF3) helicase toward the C-terminus, as well as ATPase activity. It binds to ssDNA, RNA, and site-specifically on duplex DNA at reiterations of the tetranucleotide sequence 5′-ACCA-3′1–3. These sequences are present in the viral replication origin sites and repeated at multiple sites throughout the genome in more or less degenerative forms.
NS1 nicks the covalently-closed right-end telomere via a transesterification reaction that liberates a basepaired 3′ nucleotide as a free hydroxyl (-OH). This reaction is assisted by a host DNA-binding protein from the high mobility group 1/2 (HMG1/2) family and is made in the replication origin, "OriR", which was created by sequences in and immediately adjacent to the right hairpin. The left-end telomere of MVM, a heterotelomeric parvovirus, contains sequences that can give rise to replication origins in higher-order duplex intermediates, but these sequences are inactive in the hairpin terminus of the monomeric molecule, so NS1 always initiates replication at the right end.
The 3′-OH that is freed by nicking acts as a primer for the DNA polymerase to start complementary strand synthesis while NS1 remains covalently attached to the 5′-end via a tyrosine residue. Consequently, a copy of NS1 remains attached to the 5′-end of all RF and progeny DNA throughout replication, packaging, and virion release. NS1 is only able to bind to this specific site by assembling into homodimers or higher order multimers, which happens naturally with the addition of adenosine triphosphate (ATP) that is likely mediated by NS1's helicase domain. "In vivo" studies have shown that NS1 can form into a variety of oligomeric states, but it most likely assembles into hexamers to fulfill the functions of both the endonuclease domain and helicase domain.
Starting from the location at the nick, it is thought that NS1 organizes a replication fork and acts as the replicative 3′-to-5′ helicase. Near its C-terminus, NS1 contains an acidic transcriptional activation domain. This domain acts to upregulate transcription starting from a viral promoter (P38 for MVM) when NS1 is bound to a series of 5′-ACCA-3′ motifs, called the "tar" sequence, positioned upstream (toward the 5′-end) of the promoter unit, and via interaction with NS1 and various transcription factors. NS1 also recruits the cellular replication protein A (RPA) complex, which is essential for establishing the new replication fork and for binding and stabilizing displaced single strands.
While NS1 is the only non-structural protein essential for all parvoviruses, some have other individual proteins that are essential for replication. For MVM, NS2 appears to reprogram the host cell for efficient DNA amplification, single-strand progeny synthesis, capsid assembly, and virion export, though it seems to lack direct involvement in these processes. NS2 initially accumulates up to three times more quickly than NS1 in the early S-phase but is turned-over rapidly by a proteasome-mediated pathway. As the infectious cycle progresses, NS2 becomes less common as P38-driven transcription becomes