Host-encoded reporters for the detection and purification of multiple enveloped viruses

Abstract

The identification of host cell factors for virus replication holds great promise for the development of new antiviral therapies. Recently, high-throughput screening methods have emerged as powerful tools to identify candidate host factors for therapeutic intervention. The development of assay systems suitable for large-scale automated screening is of particular importance for novel viruses with high pathogenic potential for which limited biological information can be developed in a short period of time. This report presents a general enzymatic reporter system for the detection and characterization of multiple enveloped viruses that does not rely on engineering of the virus. Instead, reporter enzymes are incorporated into virus particles by targeting to lipid microdomains in producer cells. The approach allows a variety of human pathogenic enveloped viruses to be detected by sensitive, inexpensive and automatable enzymatic assays. Tagged viruses can be purified quickly and efficiently by a magnetic bead-based capture method. The method allows general detection of enveloped viruses without prior reference to their sequence.

Fig. 1

Incorporation of alkaline phosphatase activity in Gag-Pol derived virus-like particles. (A) Schematic representation of AP::CD16b. (B) Incubation of 293 AP::CD16 cells with phosphatidylinositol specific phospholipase C (PI-PLC) from Bacillus thuringiensis removes AP::CD16b from the cell surface. (C) Gag-Pol derived particles release alkaline phosphatase activity in SN. 293 wild type (wt) and AP::CD16 cells were transfected with MLV Gag-Pol, supernatants were harvested 72 h later, cleared of cellular debris by filtration and analyzed for alkaline phosphatase activity. (D) AP activity is not associated with the soluble fraction after ultracentrifugation of Gag-Pol derived virus-like particles. (E) AP activity is associated with the particulate fraction after ultracentrifugation of Gag-Pol derived virus-like particles. (F) AP-CD16b is detected in the supernatants (sups) of Gag-Pol expressing cells. Lysates of cells and vesicles concentrated from cell supernatants by ultracentrifugation were resolved by SDS-PAGE and probed with mouse–anti-human (MaH)AP-specific mAb. n.s., not significant; ***p < 0.001; Student's t-test, paired, two-tailed.

Fig. 2

Incorporation of alkaline phosphatase activity in Gag-derived virus-like particles. (A) Schematic representation of AP::CD4 and FPLAP. The Flag-tag is indicated by a black box following the signal peptide (SP). TM, transmembrane domain. (B) 293ET cells were transiently co-transfected with MLV Gag-Pol and FPLAP or AP::CD4. Supernatants were harvested after 3 days and analyzed for alkaline phosphatase activity and compared to cells that were transfected with FPLAP constructs only. Results from three independent infections + standard deviation (SD) are shown. RLU, relative light units. (C) Cell lysates from A were analyzed by immunoblotting of FPLAP and AP::CD4 in the presence or absence of Gag-Pol using anti-Flag M2 antibody.

Fig. 3

Alkaline phosphatase can be incorporated into multiple enveloped viruses. (A) Vero reporter cell lines were infected with Respiratory Syncytia Virus (RSV) and assayed for alkaline phosphatase (AP) activity in supernatants after 7 days. (B) The presence of RSV was confirmed with the respiratory detection kit (Chemicon) by orange-red fluorescence specific for RSV (lower panel). (C) Vero reporter cell lines were infected with Herpes Simplex Virus 1 (HSV1) and cultured for up to 3 days. Supernatants were collected, centrifuged and analyzed for alkaline phosphatase activity. (D) Vero cells were infected with HSV1 as in (C). After 24 h, cell rounding was observed as a hallmark of efficient infection with HSV1 by light microscopy in infected cells (lower panel) compared to uninfected cells (upper panel). The presence of HSV1 was confirmed by immunoblotting with antibodies raised against HSV1 (lower panel). (E) MRC5 cells transduced with GFP or FPLAP were infected with human coronavirus 229E in 2% serum at 35 °C. After 3 days, supernatants were analyzed for alkaline phosphatase activity. (F) Cytophathic effects were visible after 3 days in cells infected with HCoV 229E (lower panel) compared to the confluent layer of GFP-positive cells in uninfected MRC5 cells (upper panel). The cells were lysed and subjected to immunoblotting with anti-HCoV antibody (Chemicon, as recommended by ATCC) that is highly specific for the 229E sub-type of HCoV, but not for OC43. A single band was detected only in cells that were infected with HCoV 229E. Results from three independent infections + standard deviation (SD) are shown. RLU, relative light units. *p < 0.05; ***p < 0.001; Student's t-test, paired, two-tailed.

Fig. 4

Incorporation of Flag-tagged alkaline phosphatase in replicating HIV. (A) 293ET cells were transfected with Flag-GRB2 (FGRB2) as control and FPLAP in combination with GFP or HIV. AP activity was determined after 2 days in SN. (B and C) Supernatants from 293ET cells expressing HIV and FPLAP or GFP were collected, pre-incubated with Flag M2 antibody and magnetic beads for 2 h at 4 °C and subsequently used for infection of CEMx174-T2 (MT2) cells. After 5 days, MT2 cells infected with plain supernatant or with captured virus were analyzed by flow cytometry for percentage of MT2 cells displaying green fluorescence (B) or mean fluorescence intensity (MFI) (C). Results from three independent infections + standard deviation (SD) are shown. RLU, relative light units. ***p < 0.001; Student's t-test, paired, two-tailed.