Extensive efforts in the development of drugs or therapeutic vaccines against HIV~1 have not been successful. Gene therapies, which involve the intracellular immunization of HIV~1 susceptible cells, face major challenges because of the diversity of HIV~1~ susceptible cells. The goal of this study is to develop an antiviral strategy against HIV~1 based on a defective interfering HIV~1 particle which interferes with the replication of wild type virus. If all elements of this strategy perform as hoped, an equilibrium of wild type and defective HIV~1 may be achieved which could potentially stabilize both virus load and T4 cell count and thereby delay the onset of AIDS. Several candidate defective interfering HIV~1 vector constructs (HD DNAs) were further evaluated: 1. The synthesis of Nef protein encoded by some of the HD DNAs was verified by immunofluorescent staining. Expression of the chimeric CD4~Env protein from two constructs which contain additional gag gene inserts was drastically reduced, suggesting that they may not be useful for the strategy. 2. HIV~1 released after cotransfection with HD DNAs appeared to be less infectious, suggesting a difference in the composition of released virus. 3. Several weeks of cocultivation of cells transfected with HIV~1 and HD DNA showed the presence of polyadenylated HD RNA in cell supernatants and the presence of excess HIV~1 RNA. 4. A recipient cell line for HD RNAs was selected. Cocultivation with HIV~1 and HD particle producing cells did not show a transfer of HD RNA at low sensitivity of the assay. 5. Packaging of an HIV~1 helper virus RNA into virus particles was detected despite the fact that the RNA lacks the """"""""essential"""""""" cis~acting HIV~1 packaging signal. 6. Electron microscopy of HD virus and HIV~1 budding from cells showed expected morphological differences in the makeup of the two envelopes. The insertion of the CD4~Env protein into the envelope has not been confirmed until now. 7. Computer modeling of the potential use of HD viruses as antivirals seem to indicate that if all elements of the defective virus were functional, these particles may be effective in lymph nodes, the reservoir of the virus, where there is a higher density of persistently infected cells. The potential future use of HD vectors as antivirals will depend on the demonstration of HD RNA transfer to HIV~1 infected cells and the efficiency of packaging of HD RNAs relative to HIV~1 RNA.
