Biphasic DNA synthesis in spumaviruses

Abstract

Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection. An early phase of viral DNA synthesis developed until 3 h postinfection, followed by a second phase, culminating 10 h postinfection. Both phases were abolished by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. Similar to other retroviruses, circular forms of viral DNA harboring two long terminal repeats were mainly found in the nucleus of infected cells. Interestingly, a fraction of these circular forms were detected in the cytoplasm and in extracellular virions, a feature shared with hepadnaviruses. Combined with packaging of both viral DNA and RNA genomes in virions, early and late reverse transcription might allow spumavirus to maximize its genome replication.

FIG.1.

Kinetics of total viral DNA and two-LTR circle synthesis in infected cells. BHK-21 cells were infected with HFV at three different MOIs (0.2, 0.06, and 0.02). (A and B) At regular intervals, viral DNA was extracted and quantified by real-time PCR. Results are given for 10⁶ cells as measured by quantification of the nuclear GAPDH gene. Quantitative values of the experiment, error bars, and standard deviations representing variations between two different quantifications of the same sample are given (B). (C) Two-LTR circles as percentages of total viral DNA during the same infection are deduced from the values given in panels A and B. (D and E) Kinetics of total viral DNA with two pairs of primers specific for gag and env. Cells were infected at an MOI of 0.2. Differences between the two curves may be explained by differences in efficiency of the two PCR experiments. Results are representative of three independent experiments.

FIG. 2.

Effect of pretreatment with AZT. The drug was added to FAB cells (BHK-21 cells harboring the β-galactosidase gene under the control of the HFV LTR) (26) at the time of infection. Under these conditions, total viral DNA (A) and two-LTR circles (B) were quantified by real-time PCR. At different times postinfection, the number of blue cells was determined following X-Gal staining (C).

FIG. 3.

Dynamics of viral DNA copy number when AZT was added 8 h postinfection and maintained in the medium. (A and B) Total viral DNA (A) and two-LTR circle copy numbers (B) for 10⁶ cells. (C) Two-LTR circles as percentages of total viral DNA.

FIG. 4.

Subcellular repartition of two-LTR junctions was measured by quantitative real-time PCR. BHK-21 cells were infected with HFV at an MOI of 0.06. (A and B) Viral DNA, total and two-LTR junctions, was quantified in cytoplasmic and nuclear fractions of infected cells (A) and in purified extracellular virions (B). (C) Quantification of GAPDH and viral forms in cell fractionation.