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.