Human B cell hybridomas were generated from viable frozen PBMC samples from subject 205F based on a protocol of EBV immortalization of peripheral blood B cells as described previously [46, 47, 48] . This protocol includes immortalization of pre-selected (CD22+, IgM2, IgD2, IgA2) memory B cell populations that are cultured in medium supplemented with immunostimulatory CpG sequences and irradiated allogeneic PBMC, as described in [49, 50, 51] . EBV-transformed B cell cultures were screened for Mabs that neutralized the 205F 0-month Env and that bound envelope glycoproteins by ELISA [52, 53] . The method used to screen for neutralizing Mabs is a modification of the Tzm-bl (JC53-BL13) luciferase reporter cell assay originally developed by Wei et al. [15, 17, 24, 45] . EBV-transformed B cell hybridomas with neutralizing activity were cloned in the presence of CpG and irradiated PBMC. Culture supernatant was collected from the two hybridomas, 6.4C and 13.6A, and used in the neutralization studies to map targets and escape. Figure S1 Phylogenetic tree of longitudinal 185F and 205F Envs. A. A neighbor-joining tree was generated using Clustal W v. Figure S2 Amino acid sequence alignment for 205F Envs. Three 0-month Nab sensitive Envs and six Nab resistant Envs from subsequent time points were selected for study from 32 Envs. Env clones are indicated by the time point (in months), source (PB = PBMC DNA or PL = plasma), and clone number. Sequences are shown in reference to the 0-month EnvPB1.1, with amino acid differences indicated by the letter, and deleted residues indicated by a dot. Domains that were transferred into the 0-month Env to create chimeras are as follows: V1V5 (blue, gray and green; HXB2 nt 6557 to 7634), V1V2 (blue; HXB2 nt 6557 to 6876), V3V5 (green; HXB2 nt 7110 to 7634). Major Env domains are indicated above the region, and the a2 helix is underlined. Two potential N-linked glycan addition sites of interest in V1 and V2 (NXS or NXT where X is any residue but proline) are highlighted yellow. Found at: doi:10.1371/journal.ppat.1000594.s002 (0.37 MB PDF) Figure S3 Amino acid sequence alignment for 185F Envs. Two 0-month Nab sensitive Envs and ten Nab resistant Envs from subsequent time points were selected for study from 58 Envs. Env clones are indicated by time point (in months), the source (PB = PBMC DNA or PL = plasma), and clone number. Sequences are shown in reference to the 0-month Env PL3.1, with amino acid differences indicated by the letter, and deleted residues indicated by a dot. Domains that were transferred into the 0month Env to create chimeras are as follows: V1V5 (blue, gray, and green; HXB2 nt 6577 to 7646), V1V2 (blue; HXB2 nt 6577 to 6810), V3V5 (green; HXB2 nt 7119 to 7646), gp41 ectodomain (yellow; HXB2 nt 7755 to 8270). Found at: doi:10.1371/journal.ppat.1000594.s003 (0.50 MB PDF) Figure S4 The major determinants of Nab resistance in 185F Envs change over time. Neutralization of 185F parental and chimeric Env pseudoviruses was evaluated using longitudinal plasma samples that are indicated in each panel. Each plasma sample was contemporaneous with the Nab resistant Env, and neutralization sensitivity was evaluated in JC53-BL cells using luciferase as a quantitative measure. Percent virus infectivity is plotted against the reciprocal of the log10 reciprocal plasma dilution. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. All chimeras were created in the 0-month EnvPL3.1 background (red lines). In the legend, the parental Env clones are indicated, followed by each chimera that contains the indicated region from the Nab resistant Env in the 0-month Env. 'ecto' stands for the gp41 ectodomain. Found at: doi:10.1371/journal.ppat.1000594.s004 (0.22 MB PDF) Figure S5 The major determinants of Nab resistance in 205F Envs are in V1V2. Neutralization of 205F parental and chimeric Env pseudoviruses was evaluated using longitudinal plasma samples that are indicated in each panel. Each plasma sample was contemporaneous with the Nab resistant Env, and neutralization sensitivity was evaluated in JC53-BL cells using luciferase as a quantitative measure. Percent virus infectivity is plotted against the reciprocal of the log10 reciprocal plasma dilution. Error bars represent the standard deviation of at least two independent experiments using duplicate wells. All chimeras were created in the 0-month EnvPL6.3 background (red lines). In the legend, the parental Env clones are indicated, followed by each chimera that contains the indicated region from the Nab resistant Env in the 0-month Env. Found at: doi:10.1371/journal.ppat.1000594.s005 (0.14 MB PDF) Figure S6 Highlighter plot showing nucleotide mismatches in the gp120 V1V4 region for 205F Envs. Single genome amplified, uncloned V1V4 sequences derived from the 1-Mar-03 sample (n = 21) or the 27-Mar-03 sample (n = 31) from 205F and V1V4 sequences from single genome amplified cloned Envs from the 27-Mar-03 sample (n = 5) were subjected to Highlighter analysis (www.hiv.lanl.gov). Ticks represent mismatched bases compared to the master sequence listed at the top. The three 0-month Envs that were analyzed for neutralization by 6.4C and 13.6A in Fig. 11 are boxed in red. PB = uncultured PBMC DNA; PL = plasma. The G.A mutation (diamonds) located near the beginning of the sequence represents the loss of a potential N-gly site in V1 that tracks with resistance against 13.6A. Found at: doi:10.1371/journal.ppat.1000594.s006 (0.11 MB PDF) Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE BACKGROUND: UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family. PRINCIPAL FINDINGS: We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements. CONCLUSIONS: The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. Sialic acids are Nor Osubstituted terminal monosaccharides with a nine-carbon backbone highly expressed on eukaryotic cell surfaces [1] . Sialylation of glycoproteins and glycolipids modulates a wide range of biological and pathological events including early development [2] , tumorigenesis [3] , viral and bacterial infection, and immunity [4, 5] . In vertebrate systems, N-acetylneuraminic acid (Neu5Ac) is the metabolic precursor of all known naturally occurring sialic acids [6] . Neu5Ac is synthesized in the cytosol from UDP-N-acetylglucosamine (UDP-GlcNAc) by four consecutive reactions; and UDP-GlcNAc is a derivative of fructose-6phosphate and the end-product of the hexosamine biosynthesis pathway (Figure 1 ).