Infectivity in the lack of lectin was set as 100%, and error bars indicate SD. which remain noncovalently associated (4). A hallmark of Env is its heavy glycosylation: Env is extensively modified by N-glycans, and its glycan coat protects underlying epitopes from neutralizing antibodies (5, 6). Moreover, the Env glycans are bound by immune cell lectins (7), and these interactions are believed to modulate the efficiency of mucosal transmission by shaping immune responses (8) and by targeting the virus for either transmission or degradation (9,C11). N-glycosylation commences in the endoplasmic reticulum (ER) with the en bloc transfer of a mannose-rich precursor oligosaccharide onto a protein. These oligosaccharides are trimmed by ER and 0.05; **, 0.01; ***, 0.001. SIVmac239/316 Env produced in GnTI? cells harbors exclusively high-mannose type N-glycans. We focused our subsequent analyses on SIVmac239/316 Env because this virus showed the most profound reduction in infectivity when produced in GnTI? cells. First, we confirmed that virions generated in GnTI? cells (termed GnTI-SIV) and control cells (termed wt SIV) are differentially glycosylated. For this, viral particles were concentrated by centrifugation through a sucrose cushion and analyzed by Western blotting JIB-04 (Fig. 2A). Both virus preparations contained more gp160 than proteolytically processed gp120. This effect was not unexpected since earlier data demonstrated reduced cleavage efficiency when large amounts of Env are produced (29). Moreover, gp160/gp120 proteins of GnTI-SIV migrated faster than their counterparts of wt SIV (Fig. 2A), in keeping with incorporation of high-mannose type N-glycans, which exhibit a lower molecular JIB-04 weight than hybrid- or complex-type N-glycans. Indeed, digest with endo–agglutinin (GNA) than wt SIV (Fig. 3A). In contrast, infectivity of GnTI-SIV and wt SIV was slightly and comparably augmented by agglutinin (UEA), which recognizes fucose (Fig. 3A). Thus, GnTI-SIV is more readily inhibited by mannose-specific lectins than wt SIV, in line with the exclusive modification of JIB-04 GnTI-SIV with high-mannose type N-glycans. Open in a separate window FIG 3 GnTI-SIV is more sensitive to neutralization by soluble, JIB-04 mannose-specific lectins and is better transmitted by mucosal lectins than wt SIV. (A) Infectivity-normalized stocks of wt SIV, GnTI-SIV, HIV-1 NL4-3 (positive control), and HIV-1 NL4-3 pseudotyped with the G protein of vesicular stomatitis virus (VSV-G [negative control]) were incubated with phosphate-buffered saline (PBS) or lectins prior to infection of TZM-bl indicator cells. Input virus was removed at 5 h, and -galactosidase activity in cell lysates was analyzed at 72 h postinfection. The results of a representative experiment performed with triplicate samples are shown. Infectivity in the absence of lectin was set as 100%, and error bars indicate SD. Similar results were obtained in at least one independent experiment. (B) Parental Raji B cells (control) or Raji cells Rabbit Polyclonal to MNT engineered to express DC-SIGN (SIGN) or DC-SIGNR (SIGNR) were incubated with equal volumes of wt SIV or GnTI-SIV stocks normalized for infectivity. Unbound virus was removed by washing, the transmitter cell lines were cocultured with the CEMx174 R5 target cells, and the luciferase activity in the cell lysates was measured at 72 h post-cocultivation. The results of a single experiment performed with triplicate samples are shown and are representative of two separate experiments. Error bars indicate SD. *, 0.05; **, 0.01. c.p.s., counts per second; UEA, agglutinin; CV-N, cyanovirin-N; GNA, agglutinin. The mannose-binding lectin DC-SIGN (dendritic cell-specific ICAM3-grabbing nonintegrin, CD209) binds to HIV and SIV Env, and a number of studies (31, 32) have provided evidence that DC-SIGN on dendritic cells promotes mucosal transmission by facilitating viral transfer to adjacent T cells. We employed Raji B cells stably expressing DC-SIGN and JIB-04 the DC-SIGN-related lectin DC-SIGNR (CD209L) to analyze SIV interactions with these lectins. Virus-exposed DC-SIGN+ or DC-SIGNR+ Raji cells transmitted GnTI-SIV with significantly higher efficiency than wt SIV (Fig. 3B), demonstrating that the exclusive decoration of Env with high-mannose type N-glycans can augment viral transfer by mucosal lectins. The results obtained so far raised the question of whether the decreased viral infectivity for indicator cells or the increased capture by.