Domain Organization of Lentiviral and Betaretroviral Surface Envelope Glycoproteins Modeled with AlphaFold

The surface envelope glycoproteins of non-primate lentiviruses and betaretroviruses share sequence similarity with the inner proximal domain β-sandwich of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein that faces the transmembrane glycoprotein as well as patterns of cysteine and glycosylation site distribution that points to a similar two-domain organization in at least some lentiviruses. Here, high reliability models of the surface glycoproteins obtained with the AlphaFold algorithm are presented for the gp135 glycoprotein of the small ruminant caprine arthritis-encephalitis (CAEV) and visna lentiviruses and the betaretroviruses jaagsiekte sheep retrovirus (JSRV), mouse mammary tumor virus (MMTV) and consensus human endogenous retrovirus type K (HERV-K). The models confirm and extend the inner domain structural conservation in these viruses and identify two outer domains with a putative receptor binding site in the CAEV and visna virus gp135. The location of that site is consistent with patterns of sequence conservation and glycosylation site distribution in gp135. In contrast, a single domain is modeled for the JSRV, MMTV and HERV-K betaretrovirus envelope proteins that is highly conserved structurally in the proximal region and structurally diverse in apical regions likely to interact with cell receptors. The models presented here identify sites in small ruminant lentivirus and betaretrovirus envelope glycoproteins likely to be critical for virus entry and virus neutralization by antibodies and will facilitate their functional and structural characterization. Importance Structural information on the surface envelope proteins of lentiviruses and related betaretroviruses is critical to understand mechanisms of virus-host interactions. However, experimental determination of these structures has been challenging and only the structure of the human immunodeficiency virus type 1 gp120 has been determined. The advent of the AlphaFold artificial intelligence method for structure prediction allows high-quality modeling of the structures of small ruminant lentiviral and betaretroviral surface envelope proteins. The models are consistent with much of previously described experimental data, show regions likely to interact with receptors and identify domains that may be involved in mechanisms of antibody neutralization resistance in the small ruminant lentiviruses. The models will allow more precise design of mutants to further determine mechanisms of viral entry and immune evasion in this group of viruses and constructs for structure of these surface envelope proteins.

Download Full-text

Biased N-Glycosylation Site Distribution and Acquisition across the Antibody V Region during B Cell Maturation

The Journal of Immunology ◽

10.4049/jimmunol.1801622 ◽

2019 ◽

Vol 202 (8) ◽

pp. 2220-2228 ◽

Cited By ~ 6

Author(s):

Jana Koers ◽

Ninotska I. L. Derksen ◽

Pleuni Ooijevaar-de Heer ◽

Benjamin Nota ◽

Fleur S. van de Bovenkamp ◽

...

Keyword(s):

B Cell ◽

Glycosylation Site ◽

Cell Maturation ◽

Site Distribution ◽

B Cell Maturation ◽

V Region

Download Full-text

Host Range of Small-Ruminant Lentivirus Cytopathic Variants Determined with a Selectable Caprine Arthritis- Encephalitis Virus Pseudotype System

Journal of Virology ◽

10.1128/jvi.75.16.7384-7391.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7384-7391 ◽

Cited By ~ 19

Author(s):

Isidro Hötzel ◽

William P. Cheevers

Keyword(s):

Host Range ◽

Species Distribution ◽

North American ◽

Common Property ◽

Small Ruminant ◽

Human Cells ◽

Encephalitis Virus ◽

Envelope Glycoproteins ◽

Pseudotyped Virus ◽

Caprine Arthritis Encephalitis Virus

ABSTRACT The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, andvif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.

Download Full-text

Mutation Profiles, Glycosylation Site Distribution and Codon Usage Bias of HPV16

10.1101/2021.03.04.434005 ◽

2021 ◽

Author(s):

Zhihua Ou ◽

Wei Liu ◽

Junhua LI ◽

Hongli Du

Keyword(s):

Codon Usage ◽

Codon Usage Bias ◽

Vaccine Development ◽

Gc Content ◽

Surface Protein ◽

Nucleotide Composition ◽

Glycosylation Site ◽

Protein Glycosylation ◽

Adaptation Mechanism ◽

Site Distribution

Human papillomavirus type 16 (HPV16) is the most prevalent HPV type causing cervical cancers. Herein, using 1,597 full genomes of HPV16, we systemically investigated the mutation profiles, surface protein glycosylation sites and the codon usage bias of the eight open reading frames (ORFs) of HPV16 genomes from different lineages and sublineages. Multiple lineage- or subline-age-specific mutation sites were identified. Glycosylation analysis showed that HPV16 lineage D contained the highest number of unique potential glycosylation site in both L1 and L2 capsid protein, which might lead to their antigenic distances from other HPV16 lineages. Nucleotide composition of HPV16 showed that the overall AT content was higher than GC content at the 3rd codon position. Relatively high ENC values suggested that the HPV16 ORFs didn't have strong codon usage bias. Most of the HPV16 ORFs were mainly governed by natural selection pressure such as translational pressure, except for L2. HPV16 only shared some of the preferred codons with human, which might help reduce competition in translational resources. These findings may help increase our understanding of the heterogeneity between HPV16 lineages and sublineages, and the adaptation mechanism of HPV in human cells, which might facilitate HPV classification and improve vaccine development and application.

Download Full-text

Conservation of inner domain modules in the surface envelope glycoproteins of an ancient rabbit lentivirus and extant lentiviruses and betaretroviruses

Virology ◽

10.1016/j.virol.2007.10.038 ◽

2008 ◽

Vol 372 (1) ◽

pp. 201-207 ◽

Cited By ~ 2

Author(s):

Isidro Hötzel

Keyword(s):

Envelope Glycoproteins ◽

Surface Envelope

Download Full-text

Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex

Journal of Virology ◽

10.1128/jvi.76.15.7518-7527.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7518-7527 ◽

Cited By ~ 13

Author(s):

Christina Ochsenbauer-Jambor ◽

Sue E. Delos ◽

Mary Ann Accavitti ◽

Judith M. White ◽

Eric Hunter

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Receptor Binding ◽

Mammalian Cells ◽

Leukemia Virus ◽

Glycosylation Site ◽

Enzyme Linked Immunosorbent Assay ◽

Receptor Binding Site ◽

Avian Sarcoma

ABSTRACT We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.

Download Full-text

Molecular Cloning, Bioinformatic Analysis, and Expression ofBombyx moriLebocin 5 Gene Related toBeauveria bassianaInfection

BioMed Research International ◽

10.1155/2017/9390803 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Dingding Lü ◽

Chengxiang Hou ◽

Guangxing Qin ◽

Kun Gao ◽

Tian Chen ◽

...

Keyword(s):

Immune Response ◽

Fat Body ◽

Sequence Similarity ◽

Expression System ◽

Bioinformatic Analysis ◽

Glycosylation Site ◽

Pcr Analysis ◽

Silkworm Larvae ◽

Reading Frame ◽

Prokaryotic Expression System

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm,Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected byBeauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defendB. bassianainfection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

Download Full-text

Localization of the receptor-binding site in the collectin family of proteins

Biochemical Journal ◽

10.1042/bj2930015 ◽

1993 ◽

Vol 293 (1) ◽

pp. 15-19 ◽

Cited By ~ 46

Author(s):

R Malhotra ◽

S B Laursen ◽

A C Willis ◽

R B Sim

Keyword(s):

Binding Site ◽

Receptor Binding ◽

Sequence Similarity ◽

Lung Surfactant ◽

Protein A ◽

Surfactant Protein ◽

Receptor Binding Site ◽

Ligand Interaction ◽

N Terminus ◽

Lung Surfactant Protein

Collectin receptor (Clq receptor) has been shown to bind human Clq, mannose-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin. These ligands have a similar ultrastructure, each consisting of collagenous and globular domains, but do not show a high degree of sequence similarity. For Clq and SP-A, it has been shown that both bind to cell-surface-expressed receptor(s) via their collagenous regions and this is likely to be the case with the other ligands. Within the collagenous region, near the ‘bend’ region of the collagen triple helix in Clq, MBP and SP-A, a cluster of similar charged residues is observed. This region has been suggested to be associated with receptor binding. A similar region of charge density occurs close to the N-terminus of conglutinin. In this paper we describe a truncated form of conglutinin, which has 55 amino acids missing from the N-terminus and does not bind to the collectin receptor. The results presented here strongly indicate that receptor-ligand interaction is mediated via the N-terminal region of conglutinin, consistent with the earlier proposal for the binding site.

Download Full-text

Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein

Biochemical Journal ◽

10.1042/bj2940227 ◽

1993 ◽

Vol 294 (1) ◽

pp. 227-230 ◽

Cited By ~ 11

Author(s):

G Bellachioma ◽

J L Stirling ◽

A Orlacchio ◽

T Beccari

Keyword(s):

Cdna Clone ◽

Protein Sequence ◽

Sequence Similarity ◽

Glycosylation Site ◽

Amino Acid Sequences ◽

Mouse Macrophage ◽

Activator Protein ◽

Untranslated Sequence ◽

Gm2 Activator ◽

Gm2 Activator Protein

A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5‘ end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3′ untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr).

Download Full-text

Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140

Journal of Virology ◽

10.1128/jvi.00628-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8245-8257 ◽

Cited By ~ 71

Author(s):

Eden P. Go ◽

Alon Herschhorn ◽

Christopher Gu ◽

Luis Castillo-Menendez ◽

Shijian Zhang ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Glycosylation Site ◽

Envelope Glycoproteins ◽

Immunodeficiency Virus ◽

Membrane Envelope ◽

High Mannose ◽

Complex Glycans ◽

Hiv 1

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, which consists of the gp120 and gp41 subunits, is the focus of multiple strategies for vaccine development. Extensive Env glycosylation provides HIV-1 with protection from the immune system, yet the glycans are also essential components of binding epitopes for numerous broadly neutralizing antibodies. Recent studies have shown that when Env is isolated from virions, its glycosylation profile differs significantly from that of soluble forms of Env (gp120 or gp140) predominantly used in vaccine discovery research. Here we show that exogenous membrane-anchored Envs, which can be produced in large quantities in mammalian cells, also display a virion-like glycan profile, where the glycoprotein is extensively decorated with high-mannose glycans. Additionally, because we characterized the glycosylation with a high-fidelity profiling method, glycopeptide analysis, an unprecedented level of molecular detail regarding membrane Env glycosylation and its heterogeneity is presented. Each glycosylation site was characterized individually, with about 500 glycoforms characterized per Env protein. While many of the sites contain exclusively high-mannose glycans, others retain complex glycans, resulting in a glycan profile that cannot currently be mimicked on soluble gp120 or gp140 preparations. These site-level studies are important for understanding antibody-glycan interactions on native Env trimers. Additionally, we report a newly observedO-linked glycosylation site, T606, and we show that the fullO-linked glycosylation profile of membrane-associated Env is similar to that of soluble gp140. These findings provide new insight into Env glycosylation and clarify key molecular-level differences between membrane-anchored Env and soluble gp140.IMPORTANCEA vaccine that protects against human immunodeficiency virus type 1 (HIV-1) infection should elicit antibodies that bind to the surface envelope glycoproteins on the membrane of the virus. The envelope glycoproteins have an extensive coat of carbohydrates (glycans), some of which are recognized by virus-neutralizing antibodies and some of which protect the virus from neutralizing antibodies. We found that the HIV-1 membrane envelope glycoproteins have a unique pattern of carbohydrates, with many high-mannose glycans and also, in some places, complex glycans. This pattern was very different from the carbohydrate profile seen for a more easily produced soluble version of the envelope glycoprotein. Our results provide a detailed characterization of the glycans on the natural membrane envelope glycoproteins of HIV-1, a carbohydrate profile that would be desirable to mimic with a vaccine.

Download Full-text