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145 In Vitro Analysis of Early DNA Products of Reverse Transcription Implicates a Cellular Factor in Macaque Resistance to SIV
R. Voltan*, B. Peng, and M. Robert-Guroff
NIH, NCI, Bethesda, MD, USA
Background: During a previous vaccine study we found that macaque 359 was resistant to infection by 3 sequential mucosal SIV exposures. It showed a low level of plasma viremia only at 2 weeks post the last (intrarectal) challenge, but it never seroconverted and was able to clear the virus from the peripheral blood. Transfusion of its blood and lymph node cells to a naive macaque failed to cause infection. The animal was also resistant to in vitro infection, which was not associated with mutations in chemokine receptors (CCR3, Bonzo, BOB, CXCR4, and CCR5). To determine the nature of the resistance, we examined early events in the viral replication cycle by monitoring products of SIV reverse transcription.
Methods: CD8 depleted PBMCs from the resistant animal and 2 controls were infected in vitro with 0.5 ( 104 TCDI50 of SIVmac251. Cells were collected and DNAs extracted at 6 time points between 0 and 72 hours of infection. After standardization of DNA imput using beta-actin, 3 steps of the reverse transcription process were analysed by PCR amplification: (1) minus strand strong stop DNA, (2) DNA made after the first transfer, and (3) DNA product at the end of minus strand DNA synthesis. DNA products were quantified by phosphor-imager analysis.
Results: Cells of the 2 naive control animals showed a gradual and regular increase in concentration of RT DNA products over the time course, however cells of macaque 359 displayed a different pattern. Infection occurred and reverse transcription was appropriately initiated. Intermediate DNA products accumulated much more slowly, however, with efficiency at least 100-fold lower than the controls.
Conclusions: We conclude that the resistance of macaque 359 is due in part to a post-entry block in viral replication. An unidentified host factor seems to interfere at a very early step after virus entry, modulating the virus production, and perhaps permitting the immune system to control viremia in vivo and to clear the virus
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