These antibodies were kindly provided by both James Robinson and James Hoxie. no more sensitive to neutralization than the WT by any of the neutralizing reagents. Six of the nine mutants that did not replicate appreciably had three or more glycosylation sites eliminated; the other three replication-deficient strains involved mutation of site 15. Our results suggest that elimination of glycan attachment sites 3 and 11 enhanced the exposure of contact residues for CD4. Thus, glycans at positions 3 and 11 of SIV239 gp120 may be particularly important for shielding the CD4-binding site from antibody recognition. Vaccine-induced protection against a number of viral pathogens correlates well with neutralizing antibody (Ab) titers (2, 17, 20, 53). Some have suggested that the most effective vaccine against human immunodeficiency virus (HIV) is likely to be one capable of eliciting strong, broadly reactive neutralizing antibody responses to Env as well as broad-spectrum cellular immune responses. The poor immunogenicity of the Env spike, however, is a major obstacle to the engineering of such a vaccine. One of the features of Env that contributes to its ability to escape immune recognition is its high level of Smad3 glycosylation. HIV type 1 (HIV-1) gp120 typically contains more than 20 N-linked glycosylation sites (15) and 8 O-linked sites (3). A survey performed by Myers and Lenroot of approximately 10,000 protein sequences PSI-6206 13CD3 in the SWISS-PROT library with at least one potential N-linked glycosylation site found that the number of glycosylation sites in HIV-1RF ranked in the top 10 proteins with respect to the degree of carbohydrate modification (41). N- and O-linked glycosylation of the Env precursor protein gp160 occurs during translation. Oligomerization into trimers also occurs while the nascent protein transiently PSI-6206 13CD3 resides within the endoplasmic reticulum. The newly added carbohydrate moieties are further trimmed prior to the transport of the Env oligomer to the Golgi network, where subsequent glycan diversification occurs. Formation of the viral spike is completed within the Golgi network, where gp160 is cleaved into the PSI-6206 13CD3 surface protein gp120 and the transmembrane fusion protein gp41. These two noncovalently linked subunits form heterotrimers, which become incorporated into the viral membrane upon budding. Certain glycosylation sites on cellular or viral proteins are critical to proper protein folding, intracellular stabilization, and protection against cellular proteases (10, 13, 14, 43, 46). Loss of particular glycans can also affect viral infectivity, possibly through structural alterations that influence the PSI-6206 13CD3 ability of the glycoprotein to bind its receptors, monomer interactions within the trimer, or interactions of the surface and transmembrane fusion proteins (31, 42, 55). However, many single and multiple glycosylation sites have been shown to be dispensable to viral replication within HIV-1 gp41 (11, 23) and gp120 of both HIV-1 (4, 16, 25, 26, 34, 51) and simian immunodeficiency virus (SIV) (39, 47, 48). The dispensability of some of these N-linked glycans to viral replication and the greater sensitivity of some mutants missing glycan attachment sites to antibody-mediated neutralization (4, 16, 27, 34, 36, 45, 48, 51) suggest that these glycans may also serve to shield the spike from recognition by antibodies. Variations in the number or location of glycosylation sites, particularly within the V1/V2 and V3 loops but also on the silent face of gp120, often correlate with altered sensitivity to neutralizing antibodies (1, 7, 18, 35, 44, 50, 54). Just as the acquisition of particular N-linked sites decreases neutralization sensitivity, elimination of N-linked sites at the same or nearby locations has been shown to increase neutralization sensitivity within both HIV-1 and SIV strains, particularly in the V1/V2 and V3 loops (4, 16, 27, 34, 45, 48, 51). Less is known with regard to the effects of mutagenesis of glycosylation PSI-6206 13CD3 sites outside of the V1/V2 and V3 loops on neutralization sensitivity of SIV or HIV. To address the possibility that elimination of N-linked glycans located within the better-conserved core of gp120 might expose relatively conserved domains, we created 27 mutant strains of SIV strain 239 (SIV239) that lack one or more of 14 core N-linked glycosylation sites. The mutants that lacked N-linked glycans proximal to receptor binding sites were of primary interest. We chose to.