Accumulation of 2-long terminal repeat (LTR) circular DNA forms has been proposed as a surrogate marker of inefficient HIV-1 integration [1–3]. Few studies have compared the DNA integration rates of HIV-1 and HIV-2 . Recently, using a plasmid containing both the HIV-1 and HIV-2 DNA regions amplified during PCR quantification, we significantly detected smaller amounts of total HIV DNA in HIV-2-infected patients with CD4 above 300/μL than in their HIV-1-infected counterparts . In order to compare 2-LTR DNA production by HIV-1 and HIV-2 in vitro, relative to the total amount of HIV DNA, we synthesized a new plasmid bearing both HIV-1 and HIV-2 2-LTR sequences amplified during PCR quantification of HIV-1 and HIV-2 2-LTR DNA. These fragments span the junction between the two LTR regions of circular DNA and were synthesized with primers 2LTR-HIV1U GTAACTAGAGATCCCTCAGAC, 2LTR-HIV1L ACTAGCTTGAAGCACCATCCA and 2LTR-HIV2U TCCCATCTCTCCTAGTCGC, and 2LTR-HIV2LCTGGCCCATGWGTATARTTYTGC. This HIV-1/HIV-2 LTR plasmid was used as both a calibrator and a standard in the corresponding real-time PCR, allowing us to quantify the two viruses in strictly similar conditions. This new plasmid and the previously reported plasmid, designed for total HIV-1 and HIV-2 DNA quantification , were used here to quantify total DNA and 2-LTR DNA produced by the two viruses in human peripheral blood mononuclear cells (PBMC) and cell lines infected in vitro.
The inocula of the prototype strains HIV-1 NL4-3 and HIV-2 ROD, and a strain isolated from an antiretroviral-naive symptomatic patient infected by HIV-2 MEN, were standardized using the tissue culture infectious dose (TCID)50 method based on reverse transcriptase activity (Lenti RT activity kit, Cavidi, Sweden) on the seventh day of culture, and were calculated using the Reed-Muench method . Phytohemagglutinin-activated PBMC from five donors were infected with 500 TCID50 per million cells of HIV-1 NL4-3, HIV-2 ROD or HIV-2 MEN. Cells were pelleted after 6 h and then every 24 h until the two hundred and sixty-fourth hour, and HIV DNA was quantified by means of real-time PCR applied to 1 μg of DNA extract. Total viral DNA was quantified as previously described  and the same protocol was used for 2-LTR DNA quantification by adding Pr-2LTR-HIV1: FAM3′CTGGTGTGTAGTTCTGCCAATCAG5′TAMRA to the 2LTR-HIV1 primers for HIV-1 or Pr-2LTR-HIV2: FAM3′AGACCCTGGTCTGTTAGGACCCT5′TAMRA to the 2LTR-HIV2 primers for HIV-2 (annealing temperature 60°C).
Regardless of the PBMC's donor and the HIV-2 strain, a marked difference was found in the kinetics of HIV-1 and HIV-2 DNA production (Fig. 1). The total amount of viral DNA was lower with HIV-2 than with HIV-1, by 1.16 and 0.5 Log, respectively, between 6 and 96 h (HIV-1 NL4-3 versus HIV-2 ROD). After 96 h, total HIV-1 and HIV-2 DNA levels reached a plateau, and the difference was no longer appreciable. Whatever the PBMC donor, the amount of 2-LTR DNA was lower with HIV-2 than with HIV-1 for up to 48 h of culture, but then became higher throughout the remaining culture period (0.65 Log difference between HIV-1 NL4-3 and HIV-2 ROD at the two hundred and sixty-fourth hour). Comparison of MT4-CXCR4 and HeLa-CXCR4-CCR5 cells showed that the results were not influenced by the presence or absence of CCR5.
The time lag before 2-LTR HIV-2 DNA production was similar to that observed for total HIV-2 DNA, but the subsequent kinetic pattern was unexpected. Indeed, 2-LTR HIV-2 DNA levels, similar to 2-LTR HIV-1 DNA levels, increased rapidly but, in the plateau phase (at 168 h), 2-LTR circular forms represented only 2% of total HIV-1 NL4-3 DNA copies, compared with 21% and 18% with HIV-2 DNA ROD and MEN, respectively. This difference suggests that HIV-2 may integrate the host cell genome less efficiently than HIV-1, a possibility that would be compatible with the natural history of the two human infections.
This work was supported by ANRS. Clotilde Bertin, Fanny Lemerchain, Eléonore Wach, Sébastien Delannoy and Arnaud Fleury performed the technical part of this work. Blood was kindly provided by Etablissement de transfusion sanguine de Normandie. M.G. contributed to the virological and molecular biology studies and manuscript writing and J.B. assisted with the viral culture. J.C. Plantier is the head of the Rouen Virology Laboratory and F.S. is responsible for the study and manuscript writing.
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