Early reverse transcription is essential for productive foamy virus infection

Abstract

Background: Although viral RNA constitutes the majority of nucleic acids packaged in virions, a late occurring step of reverse transcription leads to the presence of infectious viral cDNA in foamy virus particles. This peculiarity distinguishes them from the rest of the retroviral family.

Principal findings: To evaluate the respective contribution of these viral nucleic acids in the replication of foamy viruses, their fate was studied by real-time PCR and RT-PCR early after infection, in the presence or in the absence of AZT. We found that an early reverse transcription step, which occurs during the first hours post-entry, is absolutely required for productive infection. Remarkably, sensitivity to AZT can be counteracted by increasing the multiplicity of infection (moi). We also show that 2-LTR circular viral DNA, which appears as soon as four hours post-infection, is transcriptionally competent.

Conclusion: Taken together, our data demonstrate that an early reverse transcription process, which takes place soon after viral entry, is indispensable for infectivity of FVs at low moi, when the amount of DNA-containing particles is not sufficient to lead to a productive infection. This study demonstrates a key role of the packaged viral RNA in the foamy virus infection, suggesting that the replication of this virus can be achieved by involving either viral DNA or RNA genome, depending on the condition of infection.

Figure 1. Characterization of the nucleic acid content of purified PFV particles.

Copy numbers of DNA (linear and 2-LTR circles) and RNA were determined by real-time PCR and RT-PCR. Each quantification has been performed in triplicate. However, the standard deviation is too weak to be indicated. Viral supernatants were treated with DNase or RNase. The quantification of viral RNA without reverse transcription is indicated as a control. Results were expressed in copies per ml of supernatant.

Figure 2. Kinetic analysis of PFV RNA and DNA synthesis.

A representative experiment is shown. U373-MG cells were infected in the presence or absence of AZT (100 µM). The drug was added 2 hours before infection and kept on cells during the experiment. Levels of intracellular PFV DNA and RNA were monitored during the first hours of infection. (A) Dynamics of viral RNA with and without AZT monitored by real-time RT-PCR. Synthesis and fate of total viral DNA (B), integrated DNA (C) monitored by real-time PCR. Viral RNA involved in the reverse transcription step as well as viral DNA synthesis were monitored (D). At each time point post-infection, quantification of viral RNA copies without AZT is subtracted from viral RNA copies with AZT.

Figure 3. Kinetic analysis of PFV RNA and DNA synthesis over 6 days post-infection.

U373-MG cells were infected in the presence or absence of AZT. Viral DNA (A), 2-LTR circles (B), viral RNA (C) were monitored by real-time PCR and RT-PCR. The percentage of 2-LTR circles over linear viral DNA is represented (C).

Figure 4. Transcriptional activity of 2-LTR circles.

Primers used for the quantification of U5-U3 transcript are indicated. (A). The U5, R, U3 regions of the 2-LTR circles are represented. Arrowheads indicate transcription start sites. Kinetics of total viral RNA as well as U5-U3 transcript content were evaluated during a multiple round of infection of U373-MG cells in the absence (B) or presence of AZT (C).

Figure 5. Effect of increasing MOI on infectivity under AZT treatment.

Indicator FAG cells were infected with increasing MOIs in the presence or absence of AZT at two different concentrations, 10 µM and 100 µM. Forty eight hpi, GFP expression was monitored by flow cytometry. The values represented the average of three independent experiments.