Identification of a Human Respiratory Syncytial Virus Cell Entry Inhibitor by Using a Novel Lentiviral Pseudotype System

Abstract

Lentiviral budding is governed by group-specific antigens (Gag proteins) and proceeds in the absence of cognate viral envelope proteins, which has been exploited to create pseudotypes incorporating envelope proteins from nonlentiviral families. Here, we report the generation of infectious lentiviral pseudoparticles incorporating human respiratory syncytial virus (hRSV) F protein alone (hRSV-Fpp) or carrying SH, G, and F proteins (hRSV-SH/G/Fpp). These particles recapitulate key infection steps of authentic hRSV particles, including utilization of glycosaminoglycans and low-pH-independent cell entry. Moreover, hRSV pseudoparticles (hRSVpp) can faithfully reproduce phenotypic resistance to a small-molecule fusion inhibitor in clinical development (BMS-433771) and a licensed therapeutic F protein-targeting antibody (palivizumab). Inoculation of several human cell lines from lung and liver revealed more than 30-fold differences in susceptibility to hRSVpp infection, suggesting differential expression of hRSV entry cofactors and/or restriction factors between these cell types. Moreover, we observed cell-type-dependent functional differences between hRSVpp carrying solely F protein or SH, G, and F proteins with regard to utilization of glycosaminoglycans. Using hRSVpp, we identified penta-O-galloyl-␤-D-glucose (PGG) as a novel hRSV cell entry inhibitor. Moreover, we show that PGG also inhibits cell entry of hRSVpp carrying F proteins resistant to BMS-433771 or palivizumab. This work sheds new light on the mechanisms of hRSV cell entry, including possible strategies for antiviral intervention. Moreover, hRSVpp should prove valuable to dissect hRSV envelope protein functions, including the interaction with cell entry factors. IMPORTANCELentiviral pseudotypes are highly useful to specifically dissect the functions of viral and host factors in cell entry, which have been exploited for numerous viruses. Here, we successfully created hRSVpp and show that they faithfully recapitulate key characteristics of parental hRSV cell entry. Importantly, hRSVpp accurately mirror hRSV resistance to small-molecule fusion inhibitors and clinically approved therapeutic antibodies. Moreover, we observed highly different susceptibilities of cell lines to hRSVpp infection and also differences between hRSVpp types (with F protein alone or with SH, G, and F proteins) in regard to cell entry. This indicates differential expression of host factors determining hRSV cell entry between these cell lines and highlights the fact that the hRSVpp system is useful to explore the functional properties of hRSV envelope protein combinations. Therefore, this system will be highly useful to study hRSV cell entry and host factor usage and to explore antiviral strategies targeting hRSV cell entry. H uman respiratory syncytial virus (hRSV) is an enveloped, nonsegmented, negative-strand RNA virus classified in the family Paramyxoviridae. It causes upper and lower respiratory tract disease in children and elderly and immunosuppressed persons (1-3). hRSV po...

Fusion of Enveloped Viruses in Endosomes

“…Despite the various modes of internalization, most endocytosed enveloped viruses traverse the canonical endocytic pathway and enter the cytoplasm through either early endosomes, late endosomes or endolysosomes depending on which compartment has the proper environmental cue(s) to trigger and support fusion (Figure ). While entry through endosomes does not necessarily imply a requirement for low pH for fusion , for most endocytosed enveloped viruses a major determinant of the entry site is the pH dependence of the viral fusion reaction (Table ). The pH dependence of fusion varies among enveloped viruses , and these differences can correlate with the endosomal site of fusion .…”

Lentivirus-mediated CDglyTK gene-modified free flaps by intra-artery perfusion show targeted therapeutic efficacy in rat model of breast cancer

“…Compared to other vectors, lentivectors have the potential for a greater infection efficiency and more stable host genome integration of the target gene, leading to longer therapeutic gene expression. In fact, a transferred gene was continuously expressed in mesenchymal stem cells for 2 months after injection of lentivirus-mediated enhanced GFP into the cavity of mouse femoral bone in vivo [22, 23]. The tumor inhibition effect of neural stem cells transfected with a CD/TK fusion gene using lentivectors lasted 21 days in a glioma mouse model [24], and furthermore, lentivectors have been successfully used in clinical trials for the correction of adrenoleukodystrophy, β-thalassemia, and leukemia [25].…”

Pentagalloylglucose, a highly bioavailable polyphenolic compound present in Cortex moutan, efficiently blocks hepatitis C virus entry

“…With that in mind, we selected a complex of medicinal plants that is used in TCM and Reports of the biological activity of PGG are numerous and comprise anti-oncogenic, antidiabetic, anti-inflammatory, anti-oxidant, anti-angiogenic and anti-bacterial activities (Zhang et al, 2009). As would be expected for such a bioactive compound, PGG has been described to be antiviral by inhibition of viral entry also against other viruses, including respiratory syncytial virus (RSV) (Haid et al, 2016;Yeo et al, 2002), hepatitis B virus (HBV) (Lee et al, 2006), herpes simplex virus (HSV-1) (Pei et al, 2011) and influenza A viruses (Liu et al, 2011). This points to a broad mode of action, common to enveloped viruses.…”