Characterization of the temporal accumulation of minute virus of mice replicative intermediates

Abstract

We have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65% of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10% of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90% of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer of similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.

Efficient Replication of Adeno-Associated Virus Type 2 Vectors: a cis -Acting Element outside of the Terminal Repeats and a Minimal Size

“…This hypothesis is based on their failure to observe DNA on two-dimensional agarose gels with the mobility predicted for intermediates that have been nicked in the terminal palindrome during elongation. A similar observation has been reported for MVM DNA (60). Due to the absence of the predicted DNA intermediates, Ni et al (38) suggested that terminal resolution occurs only after the completion of the elongation step.…”

“…Replicative forms that are covalently closed on one or both ends are called turnaround forms. Turnaround forms can be distinguished from extended forms by two-dimensional agarose gel electrophoresis (14,48,60), where DNA samples are subjected to electrophoresis under neutral conditions in the first dimension and under alkaline conditions in the second dimension. Monomer-length replicative-form DNA that is extended on both ends migrates as a 4.7-kb double-stranded DNA in the first dimension and denatures into 4.7-kb singlestranded DNA under alkaline conditions.…”

Parvovirus Infection Suppresses Long-Term Repopulating Hematopoietic Stem Cells

“…Rather they may correspond to uncommon DNA replicative intermediates with heterogeneous ends. Indeed, the 8-kbp species may be related to a previously described partially replicated MVM dimer (21), and the large species may be related to a concatemer resolved in two-dimensional agarose gels (65). Further research is required to determine the exact nature of these dsDNA mole- cules and the BM cell types in which they persist in healthy mice long after infectious virus clearance.…”

Minute Virus of Mice NS1 Interacts with the SMN Protein, and They Colocalize in Novel Nuclear Bodies Induced by Parvovirus Infection

“…Additionally, in NS1-transfected cells infected with wild-type MVM, transiently expressed NS1 and virus-derived NS1 are present in SAABs later than 15 h after release h after release (data not shown). Although the detailed kinetics of viral replication is incomplete, significant amounts of DNA replication, capsid production, and encapsidation appear to occur at these late times in infection when the cells have become arrested (49). APAR bodies are previously described nuclear bodies that appear immediately upon infection and that do not spatially colocalize with other known nuclear bodies such as Cajal bodies, PODs, and speckles (14).…”

“…Upon infection, parvoviruses cannot induce cells into S phase, so the virus must rely on actively dividing cells to naturally progress through the cell cycle; when the cells enter S phase, a productive, lytic infection can progress (12,13). Cells infected by autonomous parvoviruses are prevented from further progress in the cell cycle (39)(40)(41)(42) yet remain viable and sustain virus replication for many hours before they die (49).…”