Fully-spliced HIV-1 RNAs are reverse transcribed with similar efficiencies as the genomic RNA in virions and cells, but more efficiently in AZT-treated cells
© Houzet et al; licensee BioMed Central Ltd. 2007
Received: 28 March 2007
Accepted: 02 May 2007
Published: 02 May 2007
We have shown previously that HIV actively and selectively packages the spliced HIV RNAs into progeny virions. In the present study, by using a RT-QPCR and QPCR strategies, we show that spliced viral RNAs are present in infectious particles and consequently participate, along with the unspliced genomic RNA, to some of the early steps of infection such as the reverse transcription step. This work provides the first quantitative data on reverse transcription of the fully spliced viral RNAs, also called the early transcripts, in target cells but also inside virions. The latter results were obtained by measuring the natural endogenous reverse transcription activity directly on intact HIV-1 particles. Our study reveals that spliced HIV RNAs are reverse transcribed as efficiently as the genomic RNA, both in cells and virions. Interestingly, we also show that reverse transcription of spliced RNAs is 56-fold less sensitive to the inhibitor AZT than reverse transcription of the genomic RNA. Therefore, the selection mediated by inhibitors of reverse transcription used to treat patients could lead to increased representativeness of spliced forms of HIV, thus favoring recombination between the HIV DNA species and facilitating HIV recovery.
HIV particles include two-copies of full-length genomic RNA (FL RNA) which represent less than 50% of the RNA mass in virions . Indeed, HIV also packages viral spliced and cellular RNAs. Recently, in a detailed quantitative study, we showed that both singly and fully spliced viral RNAs are packaged with similar efficiencies into HIV-1 particles and by an active mechanism dependent of the FL RNA packaging . Assuming that spliced HIV RNAs are packaged in infectious particles, we postulated that they are actively involved in some of the early stages of infection such as the reverse transcription (RTion) step. By using extensive QPCR strategies (well described in ), we investigated the fate of the spliced viral RNAs in HIV-1 infected cells, more specifically during RTion. We focused on the fully spliced (FSpl) RNAs because they represent the majority of the HIV-1 spliced transcripts and because they encode the regulatory proteins Tat, Rev and Nef, required to engage infection.
Fully-spliced HIV RNAs are reverse transcribed as efficiently as the FL RNA within infected cells
Reverse transcription of spliced RNAs is less sensitive to AZT than RTion of the FL RNA
The mechanism of AZT inhibition has been well studied both in vitro  and in infected cells , showing that inhibition occurred most efficiently when the DNA products of RT reaction were long. Note that FSpl RNAs are more than 4-fold shorter than FL RNA. We also measured the effects of AZT on intermediate-length reverse-transcribed DNA products, such as the minus ssDNA and U3 cDNA (Fig. 1). As shown in Fig. 4, and similarly to "Fspl cDNA", AZT has only little effect on the synthesis of these two short RTion intermediates.
This result suggests that in the infected cells exposed to AZT, the high level of the "FSpl cDNAs" is a direct consequence of their short length.
Reverse transcription of FL and FSpl RNAs is achieved equally in intact virions
In these experiments, the pelleted virions were incubated with DNase at 37°c for 45 min. This step was crucial to reduce the pNL4-3 plasmid contamination under the level of the FL cDNA. First, we evaluated the abundance of the early reverse-transcribed intermediates, ssDNA and U3, that showed similar and significant intravirion amounts (Fig. 5A), demonstrating the presence of NERT activity in the HIV-1 progeny. Similar experiments were then conducted with primers specific for FL and FSpl cDNA and showed that FL RTion products were more abundant than "Fspl cDNA" forms. NERT efficiencies (Fig. 5B) were then calculated as ratios of intravirion DNA and intravirion RNA ((DNA/RNA) × 100). These results revealed that FSpl RNAs were reverse transcribed inside virions with similar efficiencies as FL RNA (0.06% and 0.1%, respectively) and definitively not with higher efficiencies.
The relative abundance of FSpl RTion products in infected cells (1 FSpl for 44 FL) favors the hypothesis of a possible role in early steps of infection. Interestingly, it was previously reported that the FL cDNA was expressed as a non-integrated form at the very beginning of infection to give FL RNA and spliced RNAs ([10, 11], for review see  and references herein). Our results suggest that spliced RNAs could also arise from direct expression of their non-integrated cDNAs. Such early transcriptional activity from non-integrated DNAs could be highly significant in HIV-1 infection since all protein products of the FSpl RNAs are particularly crucial to engage infection. Altogether, these packaging and RTion abilities harbored by the spliced RNAs along with their low sensitivity towards RTion inhibitor, could bring first elements to explain the recent observation of high amounts of FSpl RNAs in virions isolated from plasma of AIDS patients under highly active antiretroviral therapy . Indeed, the selection mediated by RTion inhibitors used to treat patients could lead to increased proportion of spliced forms of HIV RNAs and cDNAs, and consequently to unusual encapsidation levels. This could increase recombination events between spliced and FL DNAs . These results should also be considered with respect to gene therapy protocols that commonly used HIV-producer cells with replication-defective vectors derived from HIV . Indeed, in these systems using Psi-minus genomic RNA, encapsidation of spliced viral RNAs is drastically enhanced  and subsequent RTion of those RNAs would enable the recombination between FL and spliced forms and thus the infectious HIV recovery.
human immunodeficiency virus type 1
natural endogenous reverse transcription.
The authors thank M. Biart for the 42CD4 cell-line and laboratory members past and present, including S. Maurel and F. Smagulova for helpful discussion. We also thank C. Isel for critical reading of the manuscript. This work was supported by the CNRS, SIDACTION, ANRS and the Ministère de la Recherche (ACI) to MM. LH was supported by a fellowship from SIDACTION.
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