Inducible Expression of the ΔNGFr/F12Nef Fusion Protein as a New Tool for Anti-Human Immunodeficiency Virus Type 1 Gene Therapy

Abstract

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.

Sequences within RNA coding for HIV-1 Gag p17 are efficiently targeted to exosomes

“…HEK293, HEK293T and GPR37 cells (Sparacio et al ., ) were grown in Dulbecco's modified Eagle's medium plus 10% heat‐inactivated FCS. U937, U937 HIV‐1 (Muratori et al ., ) CEMss, CEM HN (Muratori et al ., ) and Rev‐CEM cells (i.e. indicator cells expressing GFP in the presence of both HIV‐1 Tat and Rev) (Wu et al ., ) were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FCS.…”

“…In the second generation pHR' LV, the Gag-Pol sequences are truncated at nt 663, thus leaving almost intact the whole region within the Gag p17 matrix open reading frame we identified to be involved in the HIV-1 RNA exosome incorporation. We looked at the incorporation of HIV-1-related sequences in exosomes released by TNFa-treated CEMss cells expressing the human low-affinity nerve growth factor receptor truncated in its intracytoplasmic domain (L-DNGFr) under the control of the 5′ HIV-1 LTR of a pHR' LV (CEM HN cells) (Muratori et al, 2002). Total cell RNA was analysed by Northern blot for the production of HIV-1 LTR-promoted transcripts.…”

Virological Consequences of Early Events following Cell-Cell Contact between Human Immunodeficiency Virus Type 1-Infected and Uninfected CD4 + Cells

“…U937 HIV-1 , U937puro r HIV-1 , and H9 HIV-1 are cell lines derived from the U937, U937puro r (27), and H9 cell lines, respectively, upon chronic infection with the human T-lymphotropic virus (IIIB) HIV-1 isolate. These cells, as well as the CEM GFP (15), 8E5 (12), CEM HN (26), and C8166 cell lines, were grown in RPMI medium supplemented with 10% decomplemented fetal calf serum (dFCS). Human embryonic kidney epithelial 293T cells were grown in Dulbecco's modified Eagle's medium plus 10% dFCS.…”

Production of recombinant HIV‐1 nef protein using different expression host systems: A techno‐economical comparison

“…coli ,14–20 S . cerevisiae 17, 21, Sf 9‐insect cell lines,17 and several mammalian22, 23 and human cells lines 24–26. Commercially, recombinant HIV‐1 Nef is produced for diagnostic purposes (e.g., Sigma, USA; Jena Bioscience GmbH, Germany; and Bioclone, USA).…”