462 An In Vitro Model for the Study of Latent/Persistent HIV-1 Infection in Primary CD4 Cells: Quantitation of Integrated Viral DNA Using Real-time PCR KK Koelsch*1, Y Kawano1, JK Wong1,2, DD Richman1,2, CA Spina1,2 1Univ of California at San Diego, La Jolla and 2Veterans Admin San Diego Hlthcare Sys, La Jolla, CA
Background: Viral latency is a major obstacle in the treatment of HIV infection. In vivo studies of viral and host factors that contribute to the establishment of latent HIV infection in CD4 T-cells are hindered by the low frequency of latently infected cells (1-10 per 106 circulating CD4 cells). To circumvent this problem, we used a unique long-term cell culture model to study HIV-1 infection of primary CD4 T-cells aided by the application of a recently developed real-time PCR-based method to quantitate integrated HIV DNA during in vitro infection.
Methods: Primary CD4 T-cells were FACS-sorted into CD45RA+CD62L+ naive (RA) and CD45RA- memory (RO) subsets and infected with NL4-3 virus. The infected cells, in parallel with uninfected controls, were stimulated using immobilized anti-CD3 plus anti-CD28 antibodies and cultured in the presence of selected cytokines (IL-2, IL-15, IFNgamma) for up to 21 days. During the course of culture, sequential analyses were done for cell activation, cell cycle DNA, viral DNA, and p24 production. Replication competence of the provirus and infected cells was examined by a second round of activation, which was initiated using graded doses of stimuli. Integrated HIV DNA was determined by real-time PCR using gag-specific primers following physical gel-separation of the integrated and unintegrated forms of viral DNA.
Results: With induction of proliferation, the RA+ cells switched to an activated RO+ cell phenotype. HIV infection spread within both cell cultures. After several division cycles, both the RA and RO cell cultures returned to a predominantly resting state (G0/1) by day 14. At this time, HIV survived in an integrated form in the remaining cells (e.g., 0.3-0.9 x 105 copies/500ng cellular DNA). After the second round of cell activation, high levels of HIV replication were readily induced. The infected cells proliferated to a greater degree than the uninfected control cells and appeared to have a reduced threshold for activation.
Conclusions: This long-term culture system provides a highly relevant model for the study of viral-host cell interactions in the setting of latent HIV infection in CD4 T-cells. Our cell model also has the potential to aid preclinical studies in the optimization of antiviral therapy. The real-time, PCR-based assay for HIV provirus is a sensitive tool for quantitation of integrated viral DNA for studies of viral dynamics in latent infection in vivo, as well as in vitro.
