FIG. 8.

GNA binds carbohydrate attached to Asn244, Asn460, Asn476, and Asn625 in SIVmac239. (A) SIVN1, SIVN2, and SIVN2G475E gp120 proteins have a markedly decreased GNA binding capacity compared to SIVmac239 gp120. HEK293T cells were transfected with no DNA (mock) or with the gp120 eukaryotic expression vector for SIVmac239, SIVN1, SIVN2, or SIVN2G475E. Protein released into serum-free medium was purified with the concanavalin A lectin. Equivalent amounts of protein from each sample were loaded into six wells of a high-protein-binding plate. GNA-HRP was used to probe each sample in quadruplicate. The mean GNA signal (±standard deviation) is shown in black. Sera from SIV-positive rhesus macaques were used to probe each sample in duplicate. The signal from serum binding gp120 is shown in gray. (B) Normalized GNA binding to gp120 proteins from SIVN1, SIVN2, and SIVN2G475E gp120 proteins. The SIVN2G475E variant is the SIVN2 variant with an additional replacement of glycine with glutamic acid at position 475 (see Fig. 3 and 4). GNA-HRP was used to probe each sample in quadruplicate. Sera from SIV-positive rhesus macaques were used to probe each sample in duplicate. The mean GNA signal was normalized to the mean signal from sera. The normalized GNA signal (±standard deviation) is shown. (C) SIVmac239 and SIVsmE660 gp140 proteins have a greater GNA binding capacity than SIVmac239 and SIVsmE660 gp120 proteins. Equivalent amounts of histidine-tagged SIVmac239 or SIVsmE660 gp120 or gp140 proteins were each loaded into six wells of a high-protein-binding plate. GNA-HRP was used to probe each sample in quadruplicate. A histidine-HRP antibody was used to probe each sample in duplicate. The mean GNA signal was normalized to the mean signal from the histidine tag. The normalized GNA signal (±standard deviation) is shown.