Biphasic DNA Synthesis in Spumaviruses

Human foamy virus (HFV) is the prototype of the Spumavirus genus of retroviruses. First isolated from a human cell line, it was reported later to originate from chimpanzees (9). Interestingly, spumaviruses differ from other retroviruses in many features (14). The most divergent aspect concerns the timing of reverse transcription (RT). Classically, the retrovirus replication cycle is divided into two phases. The early phase comprises the steps of entry into the cell cytoplasm, synthesis of cDNA by RT of the virion-associated RNA, nuclear transport, and integration of the viral cDNA into the host cell genome. The late phase covers viral gene expression, synthesis and processing of viral proteins, and assembly and release of virions. Although RT typically occurs during the early phase for retroviruses, it was proposed elsewhere that spumavirus cDNA was produced by RT that occurred late in the viral cycle, i.e., from newly transcribed viral RNAs, before or during virus budding (16, 25, 27). Indeed, treatment of cells with the RT inhibitor 3′-azido-3′-deoxythymidine (AZT) during the entry step was reported previously to have only a small effect on HFV replication, whereas treatment of already infected cells abolished infectivity (16, 27). Late RT provided an explanation for the massive accumulation of genomic DNA in the infected cell—by exploiting the abundance of new transcripts—and the incorporation of infectious viral DNA into up to 20% of extracellular virions (25). Such features resemble those of hepadnaviruses, typified by the hepatitis B virus, which exploits a late RT step and packages DNA in virions (13). In contrast, human immunodeficiency virus type 1 (HIV-1) particles containing full-length DNA were estimated to represent only 0.001% of extracellular virions (24). The possible existence and quantitative importance of a conventional early RT step were not documented in spumaviruses.

In the infected cell, spumaviruses accumulate multiple copies of unintegrated linear double-stranded DNA harboring a gap in the positive strand (2, 12). In most retroviruses, circular DNAs, harboring one or two long terminal repeats (LTRs), are thought to form in the nucleus, either from homologous recombination between the two LTRs or from intramolecular ligation (3, 5, 10, 18, 19, 22, 23). Accordingly, circular DNAs are usually considered a marker of nuclear import (1). However, their function during retrovirus replication, if any, remains elusive. In contrast, in hepadnaviruses, circular DNAs are essential for infectivity (21). In the case of HFV, the presence of viral circular DNAs carrying two contiguous LTRs has been reported elsewhere but their abundance was lower than that of other retroviruses (20). In addition, their subcellular distribution, as well as the kinetics of their production, remains unknown.

First, we addressed directly the kinetics of HFV DNA synthesis starting immediately after infection using a sensitive and quantitative real-time PCR assay. To quantify total viral DNA (integrated, unintegrated, linear, or circular), primers in the env (SpuInF [5′-GGA CCT GTA ATA GAC TGG AA-3′] and SpuR [5′-ATT TGC AGG TCT AAT ACT CTC C-3′]) and in the gag (GAG1 [5′-CAG GAA GTA ATG TTG AAG AA-3′] and GAG2 [5′-TCT CTC AAT TTG TCC CCA CC-3′]) genes were used. The pHSRV13 plasmid harboring the entire HFV genome (15) was used as a standard for calibration of total viral DNA quantification. In the case of two-LTR circles, the following primers, encompassing the LTR-LTR junction, were used: SpA (5′-TAG TAT AAT CAT TTC CGC TTT CG-3′) and SpF (5′-CAA TAA ACC GAC TTG ATT CGA G-3′). The pR/U3 plasmid, obtained by cloning this 413-bp PCR product in a Bluescript vector (Stratagene cloning kit), was used as a standard for calibration of two-LTR circle quantification. All amplifications were performed in a 20-μl reaction volume containing 1× Light Cycler Fast Start DNA Syber Green technique mixture (Roche Diagnostics), 3.5 mM MgCl₂, and 500 nM (each) primer.

The kinetics of viral DNA synthesis was studied in BHK-21 cells infected with HFV at different multiplicities of infection (MOIs) (Fig. 1). At an MOI of 0.2, viral DNA was detected as soon as 1.5 h postinfection, while two-LTR circles were initially detected at 3 h (Fig. 1A and B). At this MOI, viral DNA can be detected at the initial time point (t = 0), likely reflecting incoming viral DNA present in the supernatant (data not shown). Strikingly, the number of viral DNA copies increased rapidly as early as 1.5 h postinfection independently of the MOI used and stabilized around 5 h postinfection. A second phase of active viral DNA accumulation was observed, culminating around 10 h postinfection. While the biphasic kinetics was reproductively observed in different experiments, the relative importance of the two phases in DNA production was shown to depend on conditions of infection, especially MOI (Fig. 1A). The biphasic aspect of the curve is less significant for two-LTR circles (Fig. 1B). As shown in Fig. 1C, from 5 h postinfection, the percentage of two-LTR circles over total viral DNA increased to reach a peak at 9.5 h and decreased later on. Beyond 24 h, a progressive increase of linear and circular DNA was observed, probably reflecting multiple cycles of infection (Fig. 1A and B). Similar results were obtained using primers in the gag gene, confirming that total viral DNA was constituted by complete genomes (Fig. 1D and E). These observations were confirmed in human U373-MG cells (data not shown).

Early detection of viral DNA and its accumulation could result either from an early synthesis or from delayed entry of viral DNA genomes packaged into incoming virions. To address this point, BHK-21 cells were infected with HFV at an MOI of 0.2, in the presence or absence of the reverse transcriptase inhibitor AZT. At the concentration used (100 μM), AZT was previously shown to inhibit HFV RT without any cytotoxic effect (27). Whereas viral DNA was detected in infected BHK-21 cells in the absence of AZT, no viral DNA accumulation was observed in cells treated with this compound from the time of infection (Fig. 2A and B). Accordingly, treatment with AZT prevented viral production by 100- to 1,000-fold, at 48 and 72 h postinfection, respectively, as revealed by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining of β-galactosidase indicator FAB cells (26) infected at the same MOI (Fig. 2C). Pretreatment with AZT before infection, or adding this drug in the medium during infection, prevented viral DNA synthesis, indicating de novo synthesis rather than delayed entry of DNA contained in virions. Previous reports have shown that early treatment has little or no effect on HFV replication, depending on cell type and culture conditions (16, 27), in agreement with our observation that the early production step accounted only for 25% of viral DNA production under our highest-MOI conditions (Fig. 1A). This supports the interpretation that the observed phases of DNA production could correspond to the early and late RT steps. However, other hypotheses might fit these observations. For example, AZT inhibition immediately after infection might suggest the existence of a DNA production from genomic RNA (classical “early” RT) and/or RNA transcribed rapidly from viral DNA genomes either integrated or not, and immediately reverse transcribed (rapid “late” RT). In this latter case, the biphasic aspect of the curves might result from an overlap of the two processes, delay between first- and second-strand synthesis, or the overall dynamics of the involved reactions. In this context, the key question is the time required to produce new RNA transcripts after entry of viral DNA genomes. This delay is unlikely to be less than a few hours, suggesting that at least a part of the early production represents classical early RT from RNA genomes. In a second series of experiments, AZT was added 8 h postinfection. Under these conditions, an initial phase of viral DNA synthesis was observed which decreased 2 h following addition of AZT (Fig. 3A and B). Note that the percentage of two-LTR circles was more significant if AZT was maintained in the medium (Fig. 3C). This observation is consistent with a higher stability of two-LTR circles than of linear DNA, a situation recently reported for HIV-1 infection in which two-LTR circles were shown to be intrinsically stable, calling into question their use as markers of recent infection (4, 17).

In contrast to what has been reported previously for HIV-1 (4, 17), circular viral DNAs cannot be detected by Southern blotting in HFV-infected cells (data not shown). Real-time quantitative PCR allowed us to detect them and to characterize both their subcellular distribution and their ratio to total viral DNA. BHK-21 cells were infected with HFV at an MOI of 0.06, washed with phosphate-buffered saline, and lysed in a hypotonic buffer (10 mM Tris, pH 7.5; 10 mM NaCl; 0.15 mM spermine; 0.5 mM spermidine; 1 mM EDTA; 100 μg of digitonin per ml) for 5 min on ice. Pellets, constituting the nuclear fraction, were obtained after an initial centrifugation for 5 min at 1,500 rpm (Beckman), whereas the remaining supernatant was centrifuged a second time for 5 min at 13,000 rpm, leading to the cytoplasmic fraction. Real-time quantitative PCR detected two-LTR circles mainly in the nucleus of infected cells, similar to what has been reported previously for HIV-1 (7, 11, 17). However, although 97% of viral DNA was located in the nucleus, linear viral DNA, and to a lesser extent two-LTR forms, was also detected within the cytoplasm of infected cells (Fig. 4). Amplification of the nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used to quantify the number of extracted cells, with the following primers: B152 (5′-TCC CTC AAG ATT GTC AGC AA-3′) and B153 (5′-AGA TCC ACA ACG GAT ACA TT-3′). GAPDH sequences detected in the cytoplasm represented 0.1% of those detected in the nucleus; on the other hand, two-LTR circles detected in the cytoplasm represented 3% of total circular viral genomes (Fig. 4C). Taken together, these observations strongly suggest that detection of cytoplasmic two-LTR circles does not result from nuclear contamination. Two hypotheses might explain the presence of two-LTR circles in the cytoplasm. First, if circles are formed in the nucleus from imported linear viral cDNA, as usually considered, they have to be subsequently exported toward the cytoplasm. Alternatively, they might form in the cytoplasm and be imported into the nucleus where they accumulate. In this scenario, the larger amount of two-LTR circles detected in the nucleus might reflect a higher stability rather than production of circular forms in this compartment.

Finally, in reference to hepadnaviruses, we sought to determine whether circular forms were incorporated into virions. Cell-free supernatant from acutely infected BHK-21 cells was mixed with NTE buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.4], 1 mM EDTA), placed on a 20% sucrose cushion, and centrifuged at 25,000 rpm for 3 h at 4°C. Pellets were resuspended in phosphate-buffered saline, and aliquots were treated or not with 450 U of DNase I (GibcoBRL) for 5 h at 37°C as already reported (25) to minimize contamination with nonpackaged viral and cellular DNA from infected cells. The efficiency of DNase I treatment was checked in aliquots contaminated with 25,000 copies of human DNA by comparing the copy number of the β-globin gene between samples treated with DNase I and those not so treated, with commercially available materials (control kit DNA; Roche Diagnostics).

Given these stringent controls for cellular DNA contamination and DNase I efficacy, viral DNA was still detected in virions (Fig. 4B), in agreement with previous studies (25, 27). Interestingly, two-LTR circles were also detected in extracellular virions, in which they represented 0.1% of total viral DNA. This ratio did not significantly vary over time during infection (Fig. 4C). Finally, the ratio of two-LTR circles to total viral DNA was comparable in all compartments, cytoplasm, nucleus, and virions (from 0.1 to 1.2%). As we used PCR primers amplifying the env and gag regions, detection of partial RT products is unlikely, as already reported (24). Given that spumavirus capsids assemble within the cytoplasm (6) and that virus budding mainly occurs from intracellular membranes (8), it is not surprising to observe the presence of linear viral DNA and circular forms in extracellular virions.

In conclusion, our data demonstrate the existence of an early RT step during foamy virus infection as classically observed in other retroviruses.

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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.

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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).

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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.

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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.

• Copyright © 2003 American Society for Microbiology