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. 1999 May;73(5):3877-85.
doi: 10.1128/JVI.73.5.3877-3885.1999.

Activation of promoter P4 of the autonomous parvovirus minute virus of mice at early S phase is required for productive infection

L Deleu  1 , A Pujol, S Faisst, J Rommelaere
Affiliations

Activation of promoter P4 of the autonomous parvovirus minute virus of mice at early S phase is required for productive infection

L Deleu et al. J Virol. 1999 May.

Abstract

Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in trans, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G1 is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G1 were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.

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Figures

FIG. 1
FIG. 1
Cell cycle dependence of viral mRNA accumulation in MVM DNA-transfected A9 cells. A9 cells arrested in G0 by serum starvation were transfected with the viral genomic clones pMVM, pMVM-Ets, and pMVM-E2F. Serum was added to a final concentration of 20% at 24 (G2), 32 to 36 (G1/S), and 44 (G1) h posttransfection. Total RNA was extracted at 48 h posttransfection and analyzed by RNase protection assay. The localization of the radiolabeled antisense RNA probe along the viral genome, as well as the different major (M) and minor (m) mRNA species, is depicted in the right panel. The distribution of the cell population according to mitotic cycle phase is given in the bottom part.
FIG. 2
FIG. 2
Quantification of viral mRNA and DNA production in MVM DNA-transfected A9 cells growing synchronously. G0-arrested cells were transfected with pMVM, pMVM-E2F, or pMVM-Ets DNA clones containing a functional (A and B) or defective (ori−) (C and D) right-hand origin of replication. Serum was added to a final concentration of 20% at 24, 32, 36, 40, and 44 h posttransfection, corresponding to 24, 16, 12, 8, and 4 h of serum induction, respectively. Total RNA and viral DNA were isolated at 48 h posttransfection and analyzed by RNase protection and Southern blotting assays, respectively. The amounts of viral replicative-form DNA (B and D) and R1 mRNA (A and C) were quantified by means of a PhosphorImager and are expressed as ratios of the values measured at a given time post-serum induction to the corresponding values at the 4-h point. For panel B, plasmid pXNS1 drove the expression of NS-1 proteins under the control of the constitutive cytomegalovirus early promoter (24) and was introduced together with pMVM-E2F by cell cotransfection. Shown are the average values from five independent experiments (standard deviation < 10%).
FIG. 3
FIG. 3
Levels of NS-1 proteins in MVM DNA-transfected A9 cells released from serum starvation. pMVM (left)- or pMVM-E2F (right)-transfected cells were harvested at 4, 8, and 24 h after serum induction. Whole-cell protein extracts were prepared, and the NS-1 polypeptide was detected by Western blotting, with a polyclonal serum directed against the C-terminal region of the protein. M, molecular mass protein markers (in kilodaltons).
FIG. 4
FIG. 4
MVM DNA replication in cells microinjected with genomic DNA clones. Approximately 5 × 102 A9 cells from the central part of a coverslip were microinjected with the wild-type (pMVM) or mutant (pMVM-E2F) genomic DNA clone, in the absence (upper panel) or presence (lower panel) of the NS-1-expressing plasmid pXNS1. The ori− clones were deprived of a functional right-hand origin of replication. At 24 h postmicroinjection, cells were transferred onto a nitrocellulose filter and viral DNA amplification was revealed by hybridization with a radioactive probe corresponding to the VP region of MVMp DNA. The diameter of the filter is 15 mm.
FIG. 5
FIG. 5
Rescue of virus formation from transfected pMVM-E2F DNA in the presence of exogenous NS-1 proteins. A9 cells were transfected with pMVM or the mutant derivative pMVM-E2F alone or in combination with the NS-1-expressing plasmid pXNS1, incubated for 7 days, and collected. Progeny viruses were released, and their titers were determined by single-cell hybridization (A) and plaque assay (B), with A9 indicator cells. While viral DNA transfer is detected by the hybridization assay, the full infectivity of progeny virions is revealed by the plaque assay.
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