Inducible expression of the deltaNGFr/F12Nef fusion protein as a new tool for anti-human immunodeficiency virus type 1 gene therapy.

Expression of the human immunodeficiency virus type 1 (HIV-1) Nef triple mutant F12Nef strongly inhibits HIV-1 replication. We exploited such a unique feature in a novel anti-HIV-1 gene therapy design by constructing an HIV-1 Tat-defective lentivirus vector expressing the product of fusion between the low-affinity human nerve growth factor receptor truncated in its intracytoplasmic domain (deltaNGFr, NH(2) moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (deltaNGFr) and the anti-HIV-1 effector are comprised in the same fusion protein, the expression of which is targetable by HIV-1 infection. Such a vector was proved to transduce human cells efficiently and, on HIV-1 infection, it expressed high levels of the fusion protein. In addition, strong antiviral activity of the deltaNGFr/F12Nef-expressing vector was demonstrated in cell lines as well as in primary cell cultures challenged with T- or M-tropic HIV-1 isolates. Thus, the HIV-1-targetable expression of the deltaNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.

Infection of simian B lymphoblastoid cells with simian immunodeficiency virus is associated with upregulation of CD23 and CD40 cell surface markers.

Simian immunodeficiency virus (SIV) as well as human immunodeficiency virus (HIV) induce polyclonal B-cell activation and are associated with the appearance of lymphomas in their respective hosts in either the presence or the absence of other co-infecting viruses such as Epstein-Barr virus (EBV). However, the pathogenic role of these retroviruses in the development of lymphoproliferative disorders remains poorly understood. To explore the virus-B-cell interactions, two immortalized lymphoblastoid B-cell lines (SL-P1 and SL-691) were established from cynomolgus monkeys that were naturally co-infected with a simian type D retrovirus-2 (SRV-2) and with the herpes virus Macaca fascicularis (HVMF-1). We addressed their susceptibility to SIV infection and the phenotypic modifications associated with SIV infection. In response, both cell lines (1) were co-infected with HVMF-1 (latent infection) and with SRV-2 (productive infection), (2) had a transformed phenotype because they did not require exogenous growth factors, and (3) when injected into mice with severe combined immunodeficiency (SCID), generated serially transplantable tumors. The B-cell origin of SL cells was demonstrated by the presence of rearrangements of the IgH gene and by the expression of typical B-cell lineage markers, such as CD20. SL-P1 and SL-691 could be discriminated on the basis of different expressions of CD23 and CD40 and of kappa- and lambda-chains. Most importantly, SL-691 cells, but not SL-P1 cells, were susceptible to chronic noncytolytic SIV infection. This infection occurred in a CD4/CCR5/CXCR4-independent manner and was associated with the upregulated expression of CD23 and CD40 cell surface markers. In addition, CD20 expression, which progressively disappeared in SL-691 noninfected cells, was maintained in the SIV-infected counterpart. These findings support the hypothesis that SIV induce phenotypic perturbations in B cells that might eventually contribute to the development of lymphoproliferative disease.

T-tropic human immunodeficiency virus (HIV) type 1 Nef protein enters human monocyte-macrophages and induces resistance to HIV replication: a possible mechanism of HIV T-tropic emergence in AIDS.

Increasing interest has been devoted to the role that monocyte-macrophages play in the pathogenesis of AIDS. The hypothesis of an involvement in AIDS pathogenesis of human/simian immunodeficiency virus (HIV/SIV) Nef also is currently under evaluation by many investigators. The original basis of this hypothesis came from evidence that monkeys infected with a nef-deleted SIV strain failed to develop simian AIDS. Here, we show that treatment of human monocyte-derived macrophages (MDM) with recombinant HIV-1 Nef protein (rNef) induces a strong inhibition of the replication of either macrophage (M-) or dual-tropic HIV-1 strains. Through cytofluorimetric analyses, we detected internalization of FITC-conjugated rNef in MDM as early as 6 h after treatment. Confocal microscope observations demonstrated that the intracellular distribution of internalized rNef was identical to that of endogenously produced Nef. Down-regulation of the CD4 HIV receptor detected upon rNef treatment of MDM suggested that the rNef-induced HIV inhibition occurred at the virus entry step. This deduction was strengthened by the observation that CD4-independent infection was totally insensitive to rNef treatment. The specificity of all observed effects was demonstrated by immunodepletion of rNef. Finally, we showed that the resistance to HIV replication induced by rNef treatment in MDM favours the spread of T-tropic over M-tropic HIV strains in doubly infected CD4(+) lymphocyte-MDM co-cultures. We propose that extracellular Nef contributes to AIDS pathogenesis by inducing resistance to M-tropic HIV replication in MDM, thereby facilitating the switching from M- to T-tropic HIV prevalence that correlates frequently with AIDS progression.