The human immunodeficiency virus (HIV) has been shown to be etiologically associated with acquired immunodeficiency syndrome (AIDS). Genomes of several HIV isolates have been molecularly cloned and sequenced. Mutational analyses of the genome of HIVSF2 show that deletion mutants in the orf-B region (or 3' orf) yield viruses which replicate to higher titers than wild type virus. Moreover, increased amounts of unintegrated and integrated viral DNA are produced in lymphoid cultures infected with these deletion mutants. The data suggest that the orf-B gene product acts as a negative control element on HIV replication. However, the exact function(s) of the orf-B gene has not been determined. We propose to study whether the orf-B gene plays a role in governing the ability of HIV to replicate in different cell types and in the establishment of latent infection. Wild type and mutant viruses will be used to infect a variety of cell lines (e.g. neural cells, macrophages, fibroblasts) to determine whether the mutant virus displays a wider host range than wild type. The amount of viral RNA and DNA, viral proteins and infectious virus will be measured to determine at which level the orf-B gene product is exerting its regulatory function in these infected cells. Furthermore, the wild type orf-B gene will be introduced into mammalian cell vectors, expressed, and used to establish cell lines that constitutively express the orf-B gene product. Transfection of cell lines producing orf-B with the mutant genome will be performed to determine whether the trans expression of the wild type orf-B gene product down-regulates or moderates the replication of orf-B mutant virus. Finally, we will molecularly clone an HIV isolate (HIVSF247) that readily establishes a latent infection in lymphoid cell cultures. Genetic engineering techniques will be used to construct viral genomes which are recombinants between HIVSF2 (active infection) and HIVSF247 (latent infection). Infectious recombinant viruses will be tested for their ability to establish a latent infection and will enable us to determine whether orf-B or other viral genes play a role in latency and cell tropism. In addition, cell lines that constitutively express the wild type orf-B gene product will be transfected with the wild type genome to determine whether augmented expression of the orf-B gene product in these cells results in the establishment of latent infection. These proposed studies should help to define a functional role of the orf-B gene in HIV gene replication.
