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A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

ABSTRACT: Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

SUBMITTER: Esposito AM

PROVIDER: S-EPMC5019951 | biostudies-other | 2016 Mar

REPOSITORIES: biostudies-other

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A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

Esposito Anthony M AM Cheung Pamela P Swartz Talia H TH Li Hongru H Tsibane Tshidi T Durham Natasha D ND Basler Christopher F CF Felsenfeld Dan P DP Chen Benjamin K BK

Virology 20160121

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion re ...[more]

PMID: 26803470

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Project description:Ebolavirus (EBOV) and Marburgvirus (MARV) that compose the filovirus family of negative strand RNA viruses infect a broad range of mammalian cells. Recent studies indicate that cellular entry of this family of viruses requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses and others are commonly used by all viral glycoproteins. Details of this entry pathway are highlighted here. Virus entry into cells is initiated by the interaction of the viral glycoprotein(1) subunit (GP(1)) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP(1) is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP(1) are currently incomplete; however, the processed GP(1) specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP(1) processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP(2) subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.

Dataset Information

A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

Publications

A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

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