THE HUMAN IMMUNODEFICIENCY VIRUS-1 NEF GENE-PRODUCT - A POSITIVE FACTOR FOR VIRAL-INFECTION AND REPLICATION IN PRIMARY LYMPHOCYTES AND MACROPHAGES

Considerable controversy and uncertainty have surrounded the biologica l function of the Human Immunodeficiency Virus (HIV)-1 nef gene produc t. Initial studies suggested that this early, nonstructural viral prot ein functioned as a negative regulatory factor; thus, it was Proposed to play a role in establishing or maintaining viral latency. In contra st, studies in Simian Immunodeficiency Virus (SIV)(mac)-infected rhesu s monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus in fection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononucl ear cells (PBMC) with Nef(+) HIV results in enhanced replication as ev idenced by earlier gag p24 expression when compared with infections pe rformed with nef mutant viruses. Moreover, when unstimulated freshly i solated PBMC are infected with Nef(+) and Nef(-) viruses and then subs equently activated with mitogen, the Nef-induced difference in viral r eplication kinetics is even more pronounced, with the Nef(-) viruses r equiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrop hages infected with a recombinant of YU-2, a patient-derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (M OI), in that infections of unstimulated PBMC at low MOI are most depen dent upon intact nef for subsequent viral growth. We now provide evide nce that the Nef(+) HIV is more infectious than Nef(-) HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infect ion assay. In the latter case, we demonstrate that infection with equi valent doses of HIV based on virion-associated gag p24 yields five- to sixfold more infected cells if Nef(+) viral stocks were used. Further more, we find that the differential infectivity is not dependent on CD 4 down-regulation as Nef(+) virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC in fections to equivalent infectivity between that of the Nef(+) and Nef( -) viruses continues to reveal delayed viral replication in the absenc e of Nef, suggesting that secondary viral spread in PBMC is also enhan ced in Nef(+) infections. We demonstrate this directly by showing a 13 -15-fold increase in infectivity of PBMC-derived Nef(+) HIV. In summar y, these findings demonstrate a consistent positive role for the HIV-1 nef gene in promoting viral infection and replication, and suggest th at the basis for this phenotype is the increased infectivity of HIV pr oduced from cells expressing nef. These data suggest that HIV-1 Nef, a s previously shown for SIV Nef, may play an important role in establis hing a fulminant form of viral infection in vivo.