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Cell-free HIV-1Zr6 vif mutants are defective in binding to peripheral blood mononuclear cells and in internalization
T Nagashunmugam1, H M Friedman
1Department of Medicine, University of Pennsylvania, Philadelphia 19104-6073, USA.
Abstract:
The vif gene of the human immunodeficiency virus (HIV-1) is required for productive virus infection of primary blood mononuclear cells (PBMCs) and macrophages in vitro. Replication of HIV-1 vif- mutants in T-lymphoid cell lines varies and is dependent on the cell line used for virus production. To further understand the role of Vif in HIV-1 infection, we constructed to vif deletion mutants from a molecular clone derived from an African patient (HIV-1Zr6). Cell-free Zr6 vif- virus pools made from transfected rhabdomyosarcoma (RD) cells do not replicate when added to cultures of stimulated PBMCs. However, vif mutants were able to spread from transfected RD cells to PBMCs if cell-to-cell contact was permitted. By Western blot analysis, viral structural proteins expressed after transfection of RD cells by wild-type or vif mutant proviruses were indistinguishable. However, binding of vif mutants to PBMCs or to purified CD4 and virus internalization were significantly reduced when compared with wild-type virus. The defects in cell-free infection, CD4 binding, and internalization were rescued by transcomplementation using a vif expression plasmid. Our results suggest a novel level at which the HIV-1 vif gene product acts to enhance cell-free infection and indicate that vif plays an important role in promoting HIV-1 binding and internalization. Combined with the previous reports of vif's effect at other steps in infection, this suggests that vif is a pleuripotent gene product that affects multiple stages of the infective process.
Insights
The HIV-1 vif gene is crucial for virus infection. Vif enhances cell-free infection by promoting viral binding and internalization into host cells, impacting the overall infective process.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for productive infection of primary cells.
- Replication of HIV-1 vif- mutants in T-lymphoid cell lines is variable and depends on the cell line used for virus production.
Purpose of the Study:
- To investigate the role of the Vif protein in HIV-1 infection, specifically its impact on cell-free infection, viral binding, and internalization.
- To characterize vif deletion mutants derived from an African patient isolate (HIV-1Zr6).
Main Methods:
- Construction and characterization of HIV-1 vif deletion mutants.
- Assessing virus replication in primary blood mononuclear cells (PBMCs) and T-lymphoid cell lines.
- Western blot analysis to evaluate viral protein expression.
- Measuring viral binding to PBMCs and CD4+ cells, and virus internalization.
- Rescue experiments using transcomplementation with a vif expression plasmid.
Main Results:
- Cell-free HIV-1 vif- mutants produced in rhabdomyosarcoma (RD) cells did not replicate in stimulated PBMCs.
- Vif mutants could spread via cell-to-cell contact.
- Viral structural protein expression was similar between wild-type and vif mutant viruses.
- Binding of vif mutants to PBMCs and CD4+ cells, as well as virus internalization, were significantly reduced compared to wild-type virus.
- Transcomplementation with a vif expression plasmid rescued the defects in cell-free infection, CD4 binding, and internalization.
Conclusions:
- The HIV-1 Vif protein plays a critical role in enhancing cell-free infection.
- Vif is important for promoting HIV-1 binding to target cells and subsequent internalization.
- Vif acts as a pleiotropic factor, influencing multiple stages of the HIV-1 infectious cycle.