Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Conditionally replicating human immunodeficiency virus type 1 (HIV-1) variants are valuable research tools.
• A previously developed HIV-rtTA variant replicates only in the presence of doxycycline (dox).
• The nef gene was replaced by a dox-dependent transcriptional activator (rtTA), hindering Nef expression and subsequent viral replication.

Purpose of the Study:

• To restore Nef expression in the HIV-rtTA variant.
• To develop genetically stable HIV-1 tools for studying Nef function and in vivo replication.

Main Methods:

• Tested internal ribosome entry site (IRES) strategies for Nef translation.
• Employed fusion protein strategies using the FMDV 2A peptide and human ubiquitin.
• Assessed virus replication in primary T-cells and human immune system (HIS) mice.

Main Results:

• IRES-mediated translation strategies were incompatible with efficient HIV-rtTA replication.
• Fusion protein approaches successfully restored stable Nef expression.
• Nef-expressing HIV-rtTA strains showed enhanced replication in primary T-cells and HIS mice compared to Nef-deficient variants.

Conclusions:

• Fusion protein strategies enable the restoration of Nef expression in switchable HIV-1 variants.
• Restored Nef expression enhances HIV-1 replication in vitro and in vivo.
• These engineered HIV-1 variants are improved tools for HIV research, particularly for studying Nef's role.