Related items
Showing items related by title, author, creator and subject.
-
Targeting N-glycan cryptic sugar moieties for broad-spectrum virus neutralization: Progress in identifying conserved molecular targets in viruses of distinct phylogenetic originsIdentifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA), for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV), and human cytomegalovirus (HCMV). In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man<inf>9</inf>GlcNAc<inf>2</inf>Asn (Man9)-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn). These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation. Copyright 2015 by the authors.
-
The Envelope Residues E152/156/158 of Zika Virus Influence the Early Stages of Virus Infection in Human CellsEmerging infections of mosquito-borne Zika virus (ZIKV) pose an increasing threat to human health, as documented over the recent years in South Pacific islands and the Americas in recent years. To better understand molecular mechanisms underlying the increase in human cases with severe pathologies, we recently demonstrated the functional roles of structural proteins capsid (C), pre-membrane (prM), and envelop (E) of ZIKV epidemic strains with the initiation of viral infection in human cells. Specifically, we found that the C-prM region contributes to permissiveness of human host cells to ZIKV infection and ZIKV-induced cytopathic effects, whereas the E protein is associated with viral attachment and early infection. In the present study, we further characterize ZIKV E proteins by investigating the roles of residues isoleucine 152 (Ile152), threonine 156 (Thr156), and histidine 158 (His158) (i.e., the E-152/156/158 residues), which surround a unique N-glycosylation site (E-154), in permissiveness of human host cells to epidemic ZIKV infection. For comparison purpose, we generated mutant molecular clones of epidemic BeH819015 (BR15) and historical MR766-NIID (MR766) strains that carry each other's E-152/156/158 residues, respectively. We observed that the BR15 mutant containing the E-152/156/158 residues from MR766 was less infectious in A549-Dual™ cells than parental virus. In contrast, the MR766 mutant containing E-152/156/158 residues from BR15 displayed increased infectivity. The observed differences in infectivity were, however, not correlated with changes in viral binding onto host-cells or cellular responses to viral infection. Instead, the E-152/156/158 residues from BR15 were associated with an increased efficiency of viral membrane fusion inside infected cells due to conformational changes of E protein that enhance exposure of the fusion loop. Our data highlight an important contribution of E-152/156/158 residues to the early steps of ZIKV infection in human cells.
-
Study of the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus LTR directed gene expression using the green fluorescent protein (GFP) as the reporterUsing enhanced green fluorescence protein (EGFP-1), a transient transfection reporter system was established to monitor the transcriptional activity of the long terminal repeats (LTR) of several primary strains of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus SIVmac239. After transient transfection of HeLa cells with variant HIV-1 LTR-EGFP-1 constructs, we scanned the cell culture using fluorescent activated cell sorter (FACS). Using FACS it was possible to simultaneously estimate for transfection efficiency and to quantitatively determine the fluorescence intensity of the transfected population. Data showed that expression of EGFP-1 was DNA dose dependent. FACS enabled the visualization of heterogeneity in the level of reporter gene expression in a transiently transfected population. The distribution of the fluorescent intensity of transfected cells was treated as a frequency distribution, and different statistical estimators were used to quantitate the amount of EGFP-1 expression. The sensitivity of the system was compared to that of chloramphenicol acetyl transferase (CAT) assay. In HeLa cells the EGFP-1 reporter assay was more sensitive than CAT assay in the absence of Tat transactivation. In HeLa cells using the EGFP-1 reporter system it was possible to demonstrate that the two coding exons of SIVmac Tat was required for optimal transactivation of the SIVmac LTR, while the first coding exon of HIV-1 Tat was sufficient to transactivate both the HIV-1 and the SIVmac LTR. However, in T cells lines while we observed low levels of transactivation from the HIV-1 and SIVmac LTR by HIV-1 Tat, we were unable to detect enhanced expression of EGFP-1 in the presence of the two coding exons of SIV mac Tat. Since the EGFP-1 reporter assay is not an enzyme based assay, in the absence of signal amplification the sensitivity of the assay in T cell lines was poor. Hence, it was difficult to detect small differences in Tat activated gene expression in the presence of either the single coding exon or both the coding exons of SIVmac Tat in T cell lines.