Enzyme Digests Eliminate Nonfunctional Env from HIV-1 Particle Surfaces, Leaving Native Env Trimers Intact and Viral Infectivity Unaffected

Abstract

HIV-1 viruses and virus-like particles (VLPs) bear nonnative "junk" forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or nearcomplete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer VLPs" may be useful neutralizing antibody immunogens.

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

“…Stability and Sensitivity of Deglycosylated gp140 Trimers to Proteolysis-Glycans can shield the underlying protein domains from proteases, so a deglycosylated protein might be atypically vulnerable to proteolytic digestion (23,42,72). We therefore compared the sensitivities of purified, deglycosylated, and mock-treated SOSIP.681 GnTϪ/Ϫ trimers to different concentrations (0.001-1 g/ml) of three common serine proteases: proteinase K, trypsin, and chymotrypsin.…”

“…The considerable heterogeneity of the glycan structures present on HIV-1 Env compromises the efficiency of various deglycosylation strategies (17,42). In previous studies using monomeric gp120 or virions, many different glycosidases were tested under a range of experimental conditions, some of which are harsh enough to probably be incompatible with Env trimer stability (8).…”

“…To date, no crystal structure of an Env trimer has been obtained, in part because it has not been possible to produce stable and soluble deglycosylated Env trimers. Deglycosylating trimeric Env on virions has proven problematic, probably because steric factors restrict access of glycosidases to their substrates (17,42). Partially deglycosylated, uncleaved gp140 trimers have recently been described (43).…”

Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

“…nantly Endo H-sensitive gp160 species in pseudoviral particles (47). This form of gp160 may bypass the normal processing pathway through the Golgi and be trafficked directly from the ER to the cell membrane (52,53).…”

GIBaren aurkako txertoaren bila

“…Izan ere, mintzean Env ezfuntzional ugari adierazten dira. Hala ere, ikusi da arazo hau konpon daitekeela Endo H, kimotripsina eta halako entzimen bitartez Env ezfuntzionalak mintzetik kenduz eta, aldiz, Env funtzionalak mintzean mantenduz [44]. Modu honetan erantzun immunea trimero funtzionaletara bideratuta, nabarmen murriztu ahal da antigorputz ezneutralizatzaileen kopurua [45].…”