Abstract
BackgroundThe ability of Human Immunodeficiency Virus (HIV) to persist in the body has proven to be a long-standing challenge to virus eradication. Current antiretroviral therapy cannot selectively destroy infected cells; it only halts active viral replication. With therapeutic cessation or interruption, viral rebound occurs, and invariably, viral loads return to pre-treatment levels. The natural reservoirs harboring replication-competent HIV-1 include CD4 T cells and macrophages. In particular, cells from the macrophage lineage resist HIV-1-mediated killing and support sustained viral production. To develop a complementary strategy to target persistently infected cells, this proof-of-concept study explores an HIV-1 Rev-dependent lentiviral vector carrying a bacterial hemolysin, anthrolysin O (anlO) from Bacillus anthracis, to achieve selective killing of HIV-1- infected cells.ResultsWe demonstrate that in the Rev-dependent lentiviral vector, anlO expression is exclusively dependent on Rev, a unique HIV-1 protein present only in infected cells. Intracellular expression and oligomerization of AnlO result in membrane pore formation and cytolysis. We have further overcome a technical hurdle in producing a Revdependent AnlO lentivirus, through the use of β-cyclodextrin derivatives to inhibit direct killing of producer cells by AnlO. Using HIV-1-infected macrophages and T cells as a model, we demonstrate that this Rev-dependent AnlO lentivirus diminishes HIV-1- positive cells.ConclusionThe Rev-dependent lentiviral vector has demonstrated its specificity in targeting persistently infected cells. The choice of anlO as the first suicidal gene tested in this vector is based on its cytolytic activity in macrophages and T cells. We conclude that Rev-regulated expression of suicidal genes in HIV-1-positive cells is possible, although future in vivo delivery of this system needs to address numerous safety issues.
Highlights
The ability of Human Immunodeficiency Virus (HIV) to persist in the body has proven to be a long-standing challenge to virus eradication
Using the green fluorescent protein (GFP) as a reporter gene, we have demonstrated that this Revdependent vector, when assembled into a viral particle and delivered into target cells, is fully dependent on HIV with no detectable background expression in uninfected cells [34,36]
To demonstrate the operation of the internal ribosome entry site (IRES), we inserted both the E. coli lacZ and the GFP gene into a single vector. This construct, pNL-LacZ-GFP-Rev responsive element (RRE)-SA (Fig. 1A), was cotransfected with an HIV-1 helper plasmid, pCMVΔ8.2 (Fig. 1B) [41], which provides both Tat and Rev to mediate the expression of LacZ and GFP in the same cell
Summary
The ability of Human Immunodeficiency Virus (HIV) to persist in the body has proven to be a long-standing challenge to virus eradication. The success of highly active antiretroviral therapy (HAART), marked by the drastic reduction of plasma viremia and restoration of certain immune functions [13], led initially to speculation of disease eradication in 2 to 3 years [4,5] This original optimism was soon dampened by the realization that persistence of viral reservoirs would make it extremely difficult, if not impossible, to eradicate HIV-1 [6,7,8,9,10,11]. Antiretroviral drugs are poorly efficacious against chronically infected macrophages [22,23,24] These features suggest that macrophages are a major viral reservoir in the body [10,11,17,22,25] and an attractive target for testing alternative therapies aimed at eradicating HIV reservoirs
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