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Abstract
The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1-V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFPOPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies.
Highlights
Validation of the trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) quaternary structure is required
Insertional mutagenesis analysis showed that the V4 and V5 loops of HIV-1 Env were tolerant to insertion of GFP
GFP Insertional Mutagenesis against HIV-1 Env—We probed the structure of gp120 by inserting GFPOPT into the variable loops of HIV-1 HXB2 Env (Table 1)
Summary
Validation of the trimeric HIV-1 envelope glycoprotein (Env) quaternary structure is required. Recent structural analyses have provided quaternary structural models for gp120/gp trimers, including the variable loops (V1–V5) of gp120. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies. We evaluated the effects of the insertion of GFPOPT or other tag proteins into gp120 variable loops on the expression, intracellular distribution, processing, and fusogenicity of this subunit. The V4 and V5 loops were more tolerant of the insertion, and GFPOPT insertion therein could be used for image analyses
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