Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases

Abstract

Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations of N-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-␤-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and ␤-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-␤-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.

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

“…Deglycosylation of Oligomannose-enriched gp140 TrimersEnv trimer glycans on SIV virions are more resistant to glycosidase digestion than those on gp120 monomers, probably because steric factors impede the access of the enzymes to their substrates on the trimers (17). We therefore tested whether the native reaction conditions identified above would also work with soluble gp140 trimers.…”

“…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).…”

Macrophage-Derived Simian Immunodeficiency Virus Exhibits Enhanced Infectivity by Comparison with T-Cell-Derived Virus

“…As cell-type-specific variability in the glycosylation of HIV and SIV envelope proteins has been reported (10-12) and virion glycosylation has been shown to affect HIV and SIV infectivity (8,15), we examined differences in virion glycosylation pathways as another possible mechanism for the increased infectivity of macrophage-derived virus. Microarray analysis was used to examine differences in the expression of genes involved in glycosylation processes between uninfected primary macrophages and T cells from three donors grown under the conditions used to produce our viral stocks.…”

HIV Toxins: Gp120 as an Independent Modulator of Cell Function

“…Instead, recombinant monomeric Gp120 is believed to contain fully mature glycans Binley et al, 2010). Thus, monomeric and native, trimeric Gp120 derived from virus and infected cells, may differ in their pattern of glycosylation (Means & Desrosiers, 2000;Mizuochi 1990). A recent study of the expression of a model oligomeric Gp120 showed that N-glycosylation of varied depending on the cell type used for expression (Raska et al, 2010).…”