Article Figures & Data
Figures
- Fig. 1.
Schematic of experimental design. Stocks of virus containing both free and virion-bound gp120 were incubated with various glycosidases. After digestion, a portion of the virus was used to test for infectivity and neutralization sensitivity using CEM×174 SIV-SEAP cells. Another portion of the treated virus was subjected to high-speed centrifugation. Supernatant was removed from the resulting pellet, and the gp120 within it was immunoprecipitated with sera from SIVmac239-infected rhesus monkeys. The pellet and immunoprecipitated materials were then analyzed by SDS-PAGE and Western blotting. Ab, antibody.
- Fig. 2.
Mobilities of virion-associated, glycosidase-treated gp120 of SIVmac239 (A) and SIVmac316 (B). Virus stock containing 60 ng of p27 was incubated at 37°C with one of the various glycosidases or buffer alone for 3 h. Each of the samples was then spun for 90 min at high speed in a refrigerated microcentrifuge. Supernatant was drawn off from each sample and set aside for immunoprecipitation experiments. Each of the pellets was boiled for 5 min in loading buffer containing 10% SDS and 1% β-mercaptoethanol. Equal amounts of each sample were then subjected to SDS-PAGE and blotted onto Immobilon-P membranes. The blots were sequentially incubated with a mixture of anti-gp120 monoclonal antibodies (KK43, KK52, and KK54) and a horseradish peroxidase-conjugated anti-mouse IgG antibody. Localization of the antibodies was visualized with a SuperSignal Pico West kit (Pierce) according to the manufacturer's recommendations. Lanes: 1, mock treatment; 2, NgF-treatment; 3, N-glycosidase A treatment; 4, α-mannosidase treatment; 5, eF treatment; 6, endoglycosidase H treatment; 7, endo-β-galactosidase-treatment; 8, neuraminidase-treatment. Numbers on the left indicate the relative positions of molecular mass markers and are shown in kilodaltons. An arrow shows the location of gp120.
- Fig. 3.
Mobilities of nonpelletable, glycosidase-treated gp120 of SIVmac239 (A) and SIVmac316 (B). Supernatants from viral pellets (described in the legend to Fig. 2) were subjected to immunoprecipitation with serum from an SIVmac239-infected rhesus macaque. The samples were then electrophoresed in a 5% polyacrylamide–SDS gel, transferred to a membrane, and reacted with a mixture of anti-gp120 monoclonal antibodies KK43, KK52, and KK54. Lanes: 1, mock treatment; 2, NgF treatment; 3, N-glycosidase A treatment; 4, α-mannosidase treatment; 5, eF treatment; 6, endoglycosidase H treatment; 7, endo-β-galactosidase treatment; 8, neuraminidase treatment. Numbers on the left indicate the relative positions of molecular mass markers (in kilodaltons).
- Fig. 4.
Mobility of SIVmac239 gp120 after SDS denaturation and then glycosidase treatment. Equal amounts of SIVmac239 were subjected to high-speed centrifugation for 90 min at 4°C. The supernatant was removed, and the pellets were resuspended in 10% SDS and 1% β-mercaptoethanol. Samples were boiled for 5 min, and then excess NP-40 was added to complex the SDS. Samples were digested with various glycosidases and then electrophoresed in a 5% polyacrylamide–SDS gel, transferred to a membrane, and reacted with a mixture of anti-gp120 monoclonal antibodies KK43, KK52, and KK54. Antibody localization was visualized by Western blotting as described in Materials and Methods. Lanes: 1, mock treatment; 2, NgF treatment; 3, N-glycosidase A treatment; 4, α-mannosidase treatment; 5, eF treatment; 6, endoglycosidase H treatment; 7, endo-β-galactosidase treatment; 8, neuraminidase treatment. Numbers on the left indicate the relative positions of molecular mass markers and are shown in kilodaltons.
- Fig. 5.
Infectivity of glycosidase-treated virus. After digestion with the indicated glycosidase, SIVmac239 (A) or SIVmac316 (B) was used to infect CEM×174 SIV-SEAP cells. Twofold dilutions of virus were added to equal amounts of CEM×174 SIV-SEAP cells and then transferred to a 37°C CO2 incubator. At approximately 60 h postinfection, the cell-free supernatant was harvested and the amount of SEAP expression was measured as described in Material and Methods. The amount of SEAP expression for each dilution was used to generate a curve from which the amount of SEAP activity per nanogram of p27 was calculated. Panel A shows the average SEAP activity per nanogram of p27 SIVmac239 after treatment with the indicated glycosidase, and panel B shows infectivity of SIVmac316 after treatment with the same glycosidases. The standard error for each experiment is indicated.
- Fig. 6.
Neuraminidase treatment increases the infectivity of SIVmac239-EGFP. Equal amounts of SIVmac239-EGFP were either mock treated, treated with neuraminidase, or treated with NgF. The treated virus was used to infect CEM×174 cells (A) or 221 cells (B). At various time points postinfection, the amounts of EGFP-positive cells were quantitated by flow cytometry analysis.
- Fig. 7.
Effects of neuraminidase on cells. CEM×174 SIV-SEAP cells were treated for 6 h with 40 mU of neuraminidase, which is 10 times the largest amount of neuraminidase present during the measurement of neuraminidase-treated virus in the infectivity assay. These cells were then washed with neuraminidase-free medium and infected with either mock- or neuraminidase-treated SIVmac239. SEAP activity in the medium was assayed at approximately 60 h postinfection as described in Materials and Methods.
- Fig. 8.
Differential effects of sialic acid residues linked α2-3, α2-6, or α2-8 on viral infectivity. SIVmac239 was treated with the indicated neuraminidases as described in Materials and Methods. The treated virus was then used to infect CEM×174 SIV-SEAP cells, and the amount of SEAP activity was quantitated at approximately 60 h postinfection. The standard deviation for each experiment is indicated.
- Fig. 9.
Neutralization of viruses by sera from SIVmac239-infected rhesus macaques. SIVmac239 (A) and SIVmac316 (B), treated with the indicated glycosidases as described in Materials and Methods, were used as virus inocula in SEAP neutralization assays as described in Materials and Methods. From the neutralization curves the amount of sera, pooled from SIVmac239-infected rhesus macaques, required to neutralize 50% of the virus-induced SEAP activity as compared with nonneutralized virus was determined. These results are representative of those from three separate experiments performed with each set of viruses, and the standard deviations are indicated.
- Fig. 10.
Neutralization of viruses by sCD4. Neutralization of SIVmac239 (A) or SIVmac316 (B) was tested as described in the legend to Fig. 9, using sCD4 instead of macaque sera to neutralize viral infectivity. The concentration of sCD4 required to reduce SIVmac239- or SIVmac316-induced SEAP activity by 50% is shown. These results are representative of those from three separate experiments performed with each set of viruses, and the standard deviations are indicated.
- Fig. 11.
Mobility of gp120 from SIVmac239 grown in the presence of glycosylation inhibitors. CEM×174 cells were infected with equal amounts of SIVmac239. At 6 days postinfection, medium alone or medium containing dGJ (1 mM), swainsonine (50 μM), or DANA (1.72 mM) was added to the cultures. The medium was completely replaced with fresh inhibitor-containing medium every 24 h for 3 days. At 10 days postinfection, the cells were washed and resuspended in medium without inhibitor. Cell-free virus stocks were collected 24 h later, and the amount of p27 antigen was quantitated by enzyme-linked immunosorbent assay. Equal amounts of each virus were spun for 90 min at high speed in a microcentrifuge. The supernatant was removed, and gp120 was electrophoresed and visualized as described in the legend to Fig. 2. Lanes: 1 and 5, mock treatment; 2, dGJ treatment; 3, swainsonine treatment; 4, DANA treatment.
- Fig. 12.
Infectivity and neutralization sensitivity of SIVmac239 grown in the presence of glycosylation inhibitors. (A) Stocks of SIVmac239 grown in the presence of the indicated inhibitor were used to generate infectivity curves as described in the legend to Fig. 5. The average amounts of SEAP activity induced per nanogram of p27 of each stock from CEM×174 SIV-SEAP cells are shown, along with the standard error from three separate experiments. (B) The same stocks of virus were used as inocula for SEAP neutralization assays, and the 50% reciprocal neutralization titer of pooled SIVmac239-infected rhesus macaque sera against each stock is shown. Similar results were obtained in three separate experiments, and the standard deviations are indicated.
Tables
- Table 1.
Summary of expected and observed shifts in molecular masses of virion-associated, free, and SDS-denatured gp120 for each of the glycosidases tested
Glycosidase Specificitya Mobility (kDa) of: Predicted mobility (kDa) of gp120 after complete digestion Virion-bound gp120d Free gp120e SDS-denatured gp120f NgF R1-GlcNAc-GlcNAc⇓Asnb 110–115 80 60 60h N-glycosidase A R1-GlcNAc-GlcNAc⇓Asnc 120 120 120 60i α-Mannosidase α1-2,3,6-linked mannose 115–120 115–120 115 NPj eF R2-GlcNAc⇓GlcNAc-Asn 120 67–72 60g 67k Endoglycosidase H R3-GlcNAc⇓GlcNAc-Asn 110 75–110 97–85 NP Endo-β-galactosidase R4-(GlcNAc-Galβ1)n⇓4R5 120 110 100 NP Neuraminidase Terminal α2-3,6,8,9-Neu5Ac 120 120 120 105–91l a The arrow indicates the site of cleavage. Asn, asparagine; Gal, galactose; Neu5Ac,N-acetylneuraminic acid; R1, high-mannose, hybrid-, or complex-form oligosaccharides; R2, high-mannose, hybrid-, or biantennary-complex-form oligosaccharides; R3, high-mannose or hybrid-form oligosaccharides; R4, Gal or Neu5Ac-Gal; R5, GlcNAc or Gal.
b Blocked by core fucosylation.
c Blocked by Neu5Ac.
d Mobility of pelletable, glycosidase-treated gp120 as described in the text.
e Mobility of immunoprecipitated, nonpelletable, glycosidase-treated gp120 as described in the text.
f Mobility of SDS-denatured and then glycosidase-treated gp120 as described in the text.
g The mobility of denatured, eF-treated SIVmac239 is greater than predicted because of NgF contamination, as described in the text.
h Based on the assumption that all 24 potential N-linked carbohydrates, each contributing about 2.5 kDa, are completely removed.
i Based on the assumption that all 24 potential N-linked carbohydrates, each contributing about 2.5 kDa, are completely removed.
j NP, not possible to predict.
k Based on the assumption that all 24 potential N-linked carbohydrates are removed by cleavage in the chitobiose core, removing about 2.2 kDa each.
l Based on the assumption that all 24 potential N-linked carbohydrates have two to four sialic residues, each contributing 309 Da, that are completely removed.
