Viral Replication and Pathogenicity
Session Day and Time: Thursday 1:30 - 3:30 pm
Room: Hall A
Background: AIDS-associated R5 HIV-1 (R5-AIDS HIV-1) biological clones have greater cytopathic effects (CPE) for CD4+ thymocytes and replicate to higher levels than pre-AIDS R5 biological clones in SCID mice bearing human thymus/liver grafts (SCID-hu mice). We hypothesize that the env and nef genes encode the cytopathic determinants of R5-AIDS HIV-1.
Methods: We cloned and sequenced the V1 to V5 region of env and the nef gene from R5-AIDS and R5-pre-AIDS biological clones. We created chimeric infectious viruses containing the V1 to V5 env regions. Chimeric viruses were characterized in SCID-hu mice. Original biological clones were analyzed in a single round infection assay on thymocytes. Production of the initial reverse transcription product, strong stop DNA (SS DNA) was determined by real time PCR. Cellular tropism was determined for each clone using reporter cells bearing CD4 and one of several co-receptors. Tropism in fetal thymic organ culture was determined using a CCR5 blocking drug, TAK-779. The relative CCR5 affinity of each clone was determined by inhibition with the CCR5 antibody 2D7. The ability of each biological clone to downregulate CD4 was also assayed.
Results: The env sequence from the AIDS clone indicates it may have increased CCR5 affinity. The nef sequences from the AIDS clone show greater similarity to nef genes from rapid progressors. The recombinant R5-AIDS clone replicated to a greater extent and more consistently causes CPE in SCID-hu mice than the R5-pre-AIDS clone. Furthermore, the R5-AIDS clone showed more efficient production of SS DNA in a single round of infection. The R5 AIDS HIV-1 replicated in reporter cells expressing CCR5 and not in cells expressing any other co-receptor. In fetal thymic organ culture, the R5-AIDS HIV-1 was completely inhibited by TAK-779. Moreover, the R5-AIDS clone showed decreased sensitivity to inhibition by the CCR5 antibody 2D7. Preliminary results did not demonstrate differences in CD4 or MHC-I downregulation between clones.
Conclusions: These data indicate that the efficiency of viral entry may determine the extent of CPE mediated by R5 HIV-1 clones in the thymus. The enhanced entry efficiency shown by the R5-AIDS clone is most likely due to increased affinity for CCR5 and not due to broadened co-receptor tropism.