final program.qxd - Parallels Plesk Panel

FP 2.2
HIV-1 and HIV-2 DNA in the early phase of infection : in vitro quantification by real
time PCR using a combined HIV-1+ HIV-2 plasmid
Marie Gueudin, Jean-Christophe Plantier, Joséphine Braun, Florence Damond, and
François Simon
CHU Charles Nicolle - ROUEN CHU Bichat - PARIS
Background-Objective
HIV-2 plasma viral loads are significantly lower than those found in HIV-1 infection. Our
objective was to study the in vitro replication of HIV-1 and HIV-2 during the early stage of
infection.
Methods
To quantify HIV-1 and HIV-2 DNA production during the early phase of in vitro infection,
we produced a plasmid integrating both HIV-1 and HIV-2 DNA. Two fragments of the LTR
of HIV type 1 and type 2 were linked using overlapping common primers and integrated
in a pCR 2.1-TOPO plasmid. TCID50 of HIV-1 NL4-3 and HIV-2 ROD supernatants used
for inoculation were determined and cells were infected with 500 TCID50 of HIV-1 NL4-3
or with 500 TCID50 of HIV-2 ROD. Cultures were performed onto PBMC from 3 different
blood donors and onto MT4 -CXCR4 and Hela-P4-CCR5 cells lines. Post transcriptional
viral production was inhibited by saquinavir and DNA production kinetics were assessed
at 6, 12, 24 and 72 hours post infection. HIV-1 and HIV-2 DNA was quantified by real-time
PCR using the HIV-1 and HIV-2 plasmid as a standard. Results were expressed in Log of
HIV DNA copies/µg of total DNA extracted.
Results
Despite equivalent infectious doses, major differences in DNA production were recorded
between the both HIV types. At 6H post infection, whatever the cells, the quantity of DNA
obtained with HIV-2 ROD (2.0 log) was 2 Log lower than the quantity obtained with HIV-1
NL4-3 (4.0 Log). This sharp difference has been observed throughout the kinetic study
despite a weak HIV-2 DNA increase at 24 -72H. These results were independent from the
PBMC donors. Results were strictly similar onto MT4-P which does not express the
co-receptor CCR5 and on the cells line HeLa-P4-CCR5 which expressed both the CCR5
and the CXCR4.
Conclusions
We observed in vitro major differences between HIV-1 and HIV-2 DNA production during
the early stage of infection. Real time PCR quantification based on a single plasmid
harbouring HIV-1 and HIV-2 sequences allows us a perfect comparison between the 2 HIV
types, avoiding artificial differences in amplification. DNA synthesized depends of the HIV
RNA availability in the cell and/or of the efficiency of the reverse transcription of this viral
RNA. Our experiment does not allow us to determine if only one or the two stages are
deficient for HIV-2 but it gives a serious track to explain most completely the
physiopathology of the infection.
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