Reduction of HIV-1 Infectivity through Endoplasmic Reticulum-Associated Degradation-Mediated Env Depletion

Abstract

During the HIV-1 replicative cycle, the gp160 envelope is processed in the secretory pathway to mature into the gp41 and gp120 subunits. Misfolded proteins located within the endoplasmic reticulum (ER) are proteasomally degraded through the ER-associated degradation (ERAD) pathway, a quality control system operating in this compartment. Here, we exploited the ERAD pathway to induce the degradation of gp160 during viral production, thus leading to the release of gp120-depleted viral particles.T he precursor of the viral envelope, gp160, undergoes extensive posttranslational modification in the endoplasmic reticulum (ER) and is subsequently cleaved into two subunits, gp120 and gp41, within the Golgi apparatus. The processed Env subunits then reach the plasma membrane, where they are incorporated into the budding viral particles (1).Here, we present a novel strategy to reduce HIV-1 infectivity through the depletion of gp120 from viral particles. This approach is based on gp160 degradation during viral production obtained by using the targeted ER-associated degradation (TED) approach. This recently developed technique exploits the ER-associated degradation pathway (ERAD) machinery to promote specific downregulation of target proteins trafficking through the secretory pathway (2). TED uses chimeric molecules termed "degradins" that are characterized by two functional moieties: a target recognition moiety and a degradation-inducing moiety composed of the C-terminal fragment (amino acids [aa] 402 to 773) of the cellular ER-resident protein SEL1L. This protein is involved in the ERAD pathway by selecting misfolded proteins for retrotranslocation from the ER lumen to the cytosol for proteasomal degradation (3). SEL1L chimeras designed against selected targets have been demonstrated to specifically force the interaction of the target protein with the retrotranslocation machinery, leading to the export of the protein from the ER and its subsequent degradation in the cytosol (2).To obtain gp160-specific degradins, we prepared SEL1L chimeras containing different target recognition moieties directed against various epitopes of HIV-1 gp160. We used three singlechain antibody fragments (scFv) derived from monoclonal antibodies (MAbs): Chessie1339, obtained from the anti-gp160 hybridoma Chessie 13-39.1 (4), to produce the 1339-SEL1L degradin; and VRC01 and VRC03, derived from two broad neutralizing MAbs directed toward the CD4 binding site of gp120 (5), to produce the VRC01-SEL1L and VRC03-SEL1L degradins, respectively. A general scheme of degradin design is reported in Fig. 1A.We next tested the efficacy of the anti-gp160 degradins in 293T cells coexpressing the SEL-1L chimeras with a codon-optimized gp160. In these experiments, gp160 is expressed from a construct containing the codon-optimized sequence for gp120 (isolate JRFL, clade B) from the pSyngp120 plasmid (6) in frame with the optimized sequence for gp41 derived by gene synthesis from the same isolate. In addition, the N terminus of gp160 was modified by substituting t...

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