ABSTRACT Human immunodeficiency virus 1 (HIV-1) is a major global pathogen, with 1.7 million new infections in 2019. The HIV-1 envelope glycoprotein (Env) is the sole virus-surface antigen. Env, a trimer of gp120/gp41 dimer subunits, is densely glycosylated by N-glycans. For the gp120 subunit, N-glycans contribute over 50% of its molecular mass and form a protective surface, the so-called “glycan shield”, that interacts with cellular receptors and the immune system and protects the virus from neutralizing antibodies (nAbs). Recently, the concept of an “evolving glycan shield” has been proposed, based on the sequence analysis of immune-escape variants that have lost some potential N-glycosylation sites (NGS) and gained others. Despite significant strides in understanding the biology of HIV-1 Env, the complexity and function of N-glycans at a molecular level are poorly understood. Based on our bioinformatics and experimental studies, we recently proposed that the Env glycan shield may be defined, and analyzed, as distinct structural glycan-microdomains. Using naturally occurring Env variants of a clade B virus, we analyzed the glycans surrounding the highly conserved N262 glycan, in the so- called high-mannose patch (HMP) near the apex of the Env trimer. These HMP glycans, comprised of different combinations of 4 to 6 NGS in the proximity of N262, appear to form a glycan working unit. Based on our published results and preliminary data, we hypothesize that Env outer domain, gp120 glycan shield, has structural components that can be defined as distinct structural glycan-microdomains. We propose to test our hypothesis in different clades of HIV-1 and their immune-escape variants by assessing structural and functional impacts of a range of N-glycan combinations that comprise Env glycan-microdomains. Specifically, we propose to determine how different NGS combinations of the N262-anchored HMP-microdomain across variants and clades influence Env functions (Aim 1), to probe the structural arrangement of different N262-glycan cluster NGS combinations in the context of functional recombinant Env trimers (Aim 2), and to assess the functional impacts of other potential glycan-clusters of the HIV-1 Env glycan shield (Aim 3). To accomplish these goals, we will use a combination of functional, bioanalytical, structural bioinformatics, and molecular dynamics simulation approaches. The results of these studies will test our proposed hypothesis on the functional impact of the structural components of the glycan shield, glycan-microdomains, and will determine how the varied compositions of these microdomains affect the function of the glycan shield. These data will advance our understanding of the molecular biology of HIV-1 and the factors that impact viral transmission and persistence. Ultimately, these studies will generate new information about Env vulnerabilities that can be exploited to develop future therapeutic approaches.