Geospatial HIV-1 subtype C gp120 sequence diversity and its predicted impact on broadly neutralizing antibody sensitivity

Evolving diversity in globally circulating HIV-1 subtypes presents a formidable challenge in defining and developing neutralizing antibodies for prevention and treatment. HIV-1 subtype C is responsible for majority of global HIV-1 infections. In the present study, we examined the diversity in genetic signatures and attributes that differentiate region-specific HIV-1 subtype C gp120 sequences associated with virus neutralization outcomes to key bnAbs having distinct epitope specificities. A total of 1814 full length HIV-1 subtype C gp120 sequence from 37 countries were retrieved from Los Alamos National Laboratory HIV database (www.hiv.lanl.gov). The amino acid sequences were assessed for their phylogenetic association, variable loop lengths and prevalence of potential N-linked glycosylation sites (pNLGS). Responses of these sequences to bnAbs were predicted with a machine learning algorithm ‘bNAb-ReP’ and compared with those reported in the CATNAP database. Subtype C sequences from Asian countries including India differed phylogenetically when compared with that from African countries. Variable loop lengths and charges within Indian and African clusters were also found to be distinct from each other, specifically for V1, V2 and V4 loops. Pairwise analyses at each of the 25 pNLG sites indicated distinct country specific profiles. Highly significant differences (p<0.001***) were observed in prevalence of four pNLGS (N130, N295, N392 and N448) between South Africa and India, having most disease burden associated with subtype C. Our findings highlight that distinctly evolving clusters within global intra-subtype C gp120 sequences are likely to influence the disparate region-specific sensitivity of circulating HIV-1 subtype C to bnAbs.

Geospatial HIV-1 subtype C gp120 sequence diversity and its predicted impact on broadly neutralizing antibody sensitivity

Evolving diversity in globally circulating HIV-1 subtypes presents formidable challenge in defining and developing neutralizing antibodies for prevention and treatment. HIV-1 subtype C is responsible for majority of global HIV-1 infections. Broadly neutralizing antibodies (bnAbs) capable of neutralizing distinct HIV-1 subtypes by targeting conserved vulnerable epitopes on viral envelope protein (Env) are being considered as promising antiviral agents for prevention and treatment. In the present study, we examined the diversity in genetic signatures and attributes that differentiate region-specific global HIV-1 subtype C gp120 sequences associated with virus neutralization outcomes to key bnAbs having distinct epitope specificities. A total of 1814 full length HIV-1 subtype C gp120 sequence from 37 countries were retrieved from Los Alamos National Laboratory HIV database (www.hiv.lanl.gov). The amino acid sequences were assessed for their phylogenetic association, variable loop lengths and prevalence of potential N-linked glycosylation sites (pNLGS). Responses of these sequences to bnAbs were predicted with a machine learning algorithm ‘bNAb-ReP’ and compared with those reported in the CATNAP database. Phylogenetically, sequences from Asian countries including India clustered together however differed significantly when compared with pan African subtype C sequences. Variable loop lengths within Indian and African clusters were distinct from each other, specifically V1, V2 and V4 loops. Furthermore, V1V2 and V2 alone sequences were also found to vary significantly in their charges. Pairwise analyses at each of the 25 pNLG sites indicated distinct country specific profiles. Highly significant differences (p<0.001***) were observed in prevalence of four pNLGS (N130, N295, N392 and N448) between South Africa and India, having most disease burden associated with subtype C. Our findings highlight that the distinctly evolving clusters within global intra-subtype C gp120 sequences are likely to influence the disparate region-specific sensitivity of circulating HIV-1 subtype C to bnAbs.

Functional reprogramming of Candida glabrata epithelial adhesins: the role of conserved and variable structural motifs in ligand binding

For host–cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall-attached epithelial adhesins (Epas). Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure-based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolution.

Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.

HIV-1 Coreceptor Usage and Variable Loop Contact Impact V3 Loop Broadly Neutralizing Antibody Susceptibility

ABSTRACT In clinical trials, HIV-1 broadly neutralizing antibodies (bnAbs) effectively lower plasma viremia and delay virus reemergence. The presence of less neutralization-susceptible strains prior to treatment decreases the efficacy of these antibody-based treatments, but neutralization sensitivity often cannot be predicted by sequence analysis alone. We found that phenotypically confirmed CXCR4-utilizing strains are less neutralization sensitive, especially to variable loop 3 (V3 loop)-directed bnAbs, than exclusively CCR5-utilizing strains in some, but not all, cases. Homology modeling suggested that the primary V3 loop bnAb epitope is equally accessible among CCR5- and CXCR4-using strains, although variants that exclusively use CXCR4 have V3 loop protrusions that interfere with CCR5 receptor interactions. Homology modeling also showed that among some, but not all, envelopes with decreased neutralization sensitivity, V1 loop orientation interfered with V3 loop-directed bnAb binding. Thus, there are likely different structural reasons for the coreceptor usage restriction and the different bnAb susceptibilities. Importantly, we show that individuals harboring envelopes with higher likelihood of using CXCR4 or greater predicted V1 loop interference have faster virus rebound and a lower maximum decrease in plasma viremia, respectively, after treatment with a V3 loop bnAb. Knowledge of receptor usage and homology models may be useful in developing future algorithms that predict treatment efficacy with V3 loop bnAbs. IMPORTANCE The efficacy of HIV-1 broadly neutralizing antibody (bnAb) therapies may be compromised by the preexistence of less susceptible variants. Sequence-based methods are needed to predict pretreatment variants’ neutralization sensitivities. HIV-1 strains that exclusively use the CXCR4 receptor rather than the CCR5 receptor are less neutralization susceptible, especially to variable loop 3 (V3 loop) bnAbs in some, but not all, instances. While the inability to utilize the CCR5 receptor maps to a predicted protrusion in the envelope V3 loop, this viral determinant does not directly influence V3 loop bnAb sensitivity. Homology modeling predicts that contact between the envelope V1 loop and the antibody impacts V3 loop bnAb susceptibility in some cases. Among pretreatment envelopes, increased probability of using CXCR4 and greater predicted V1 interference are associated with faster virus rebound and a smaller decrease in the plasma virus level, respectively, after V3 loop bnAb treatment. Receptor usage information and homology models may be useful for predicting V3 loop bnAb therapy efficacy.

2D-HELS-AA MS Seq: Direct sequencing of tRNA reveals its different isoforms and multiple dynamic base modifications

We report a direct method for sequencing tRNAPhe without cDNA by combining 2-dimensional hydrophobic RNA end-labeling with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNAPhe was sequenced and the identity, location and abundance of all 11 base modifications were determined. Changes in ratios of wybutosine and its depurinated form under different conditions were quantified, pointing to the ability of our technology to determine dynamic changes of nucleotide modifications. Two truncated isoforms at 3’CCA tail of the tRNAPhe (75 nt CC, 80% and 74 nt C, 3%) were identified in addition to the 76 nt tRNAPhe with a full-length 3’CCA tail (17%). We also discovered a new isoform with A-G transitions at both the 44 and 45 positions in the tRNAPhe variable loop.One Sentence SummaryDirect 2D-HELS-AA MS Seq of tRNA reveals different isoforms and base modifications

LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

HIV-1 co-receptor usage and variable loop contact impacts V3 loop bnAb susceptibility

ABSTRACTIn clinical trials, HIV-1 broadly neutralizing antibodies (bnAbs) effectively lower plasma viremia and delay virus reemergence after antiretroviral treatment is stopped among infected individuals that have undetectable virus levels. Presence of less neutralization susceptible strains prior to treatment, however, decreases the efficacy of these antibody-based treatments. The HIV-1 envelope glycoprotein harbors extensive genetic variation, and thus, neutralization sensitivity often cannot be predicted by sequence analysis alone. Sequence-based prediction methods are needed because phenotypic-based assays are labor intensive and not sensitive. Based on the finding that phenotypically confirmed CXCR4- as compared to exclusive CCR5-utilizing strains are less neutralization sensitive, especially to variable loop 1 and 2 (V1-V2) and V3 loop bnAbs, we show that an algorithm that predicts receptor usage identifies envelopes with decreased V3 loop bnAb susceptibility. Homology modeling suggests that the primary V3 loop bnAb epitope is equally accessible among CCR5- and CXCR4-using strains although variants that exclusively use CXCR4 have V3 loop protrusions that interfere with CCR5 receptor interactions. On the other hand, homology modeling also shows that envelope V1 loop orientation interferes with V3 loop directed bnAb binding, and this accounts for decreased neutralization sensitivity in some but not all cases. Thus, there are likely different structural reasons for the co-receptor usage restriction and the differential bnAb susceptibility. Algorithms that use sequence data to predict receptor usage and antibody-envelope homology models can be used to identify variants with decreased sensitivity to V3 loop and potentially other bnAbs.AUTHOR SUMMARYHIV-1 broadly neutralizing antibody (bnAb) therapies are effective, but the pre-existence of less susceptible variants may lead to therapeutic failure. Sequence-based methods are needed to predict pre-treatment variants’ neutralization sensitivity. HIV-1 strains that use the CXCR4 as compared to the CCR5 receptor are less neutralization susceptible, especially to V1-V2 and V3 loop bnAbs. A sequence-based algorithm that predicts receptor usage can identify envelope variants with decreased V3 loop bnAb susceptibility. While the inability to utilize the CCR5 receptor maps to a predicted protrusion in the envelope V3 loop, this viral determinant does not directly influence V3 loop bnAb sensitivity. Furthermore, homology modeling predicted contact between the envelope V1 loop and an antibody also impact V3 loop bnAb susceptibility in some but not all cases. An algorithm that predicts receptor usage and homology modeling can be used to predict sensitivity to bnAbs that target the V3 loop and potentially other envelope domains. These sequence-based methods will be useful as HIV-1 bnAbs enter the clinical arena.