Phenotypic lentivirus screens to identify functional single domain antibodies

Abstract

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway.

Figure 1

Lentiviral screening approach. Alpacas are immunized with the desired antigen mix (here: inactivated influenza A virus, IAV, and vesicular stomatitis virus, VSV). After repeated immunizations, we draw a blood sample, purify lymphocytes, extract mRNA, reversely transcribe RNA to cDNA, amplify VHH coding sequences and clone them into a lentiviral vector. Alternatively, VHH coding sequences can be subcloned from an existing VHH library. 293T cells are transfected with the lentiviral library as well as packaging vectors, and lentivirus is harvested from the supernatant 2 days later. We transduce the cell line of interest (here: A549 cells) and induce VHH expression with doxycycline. Cells are then subjected to a selection assay that allows identification of cells expressing the desired VHHs (here: survival of a lethal infection with IAV or VSV). Finally, we prepared genomic DNA from selected (surviving) cells, amplified the VHH sequences by PCR and determined the VHH sequence encoded.

Figure 2

Overview of the antiviral VHH screen hits. (a) Summary of the number of transduced cells, amplified cell clones, number of confirmed hits reducing infection by more than 40% (80%), number of hits containing a single insertion, and number of different VHH clusters in the IAV and VSV screens. Amino acid sequences of the obtained anti-IAV (b) and anti-VSV (c) VHHs are presented (number independent identifications in parentheses).

Figure 3

Validation of antiviral VHHs. A549 cells or clones inducibly expressing the indicated VHHs were treated without or with 1 μg/mL doxycycline (Dox) for 24 h and subsequently infected with IAV WSN (a, c, d) or VSV Indiana EGFP (b, e, f) for 6 or 4 h, respectively. IAV-infected cells were stained for NP and VHH-HA expression; VSV-infected cells were stained for VHH-HA expression. Cells were analyzed by flow cytometry (sample histograms in (a) and (b)); the fraction of infected cells in the presence of Dox was quantified and normalized to infection in the absence of Dox. Hits from IAV screen are displayed in (c) and (d), hits from VSV screen in (e) and (f). All data is from three independent experiments ± s.e.m.

Figure 4

Identification of VHH targets. (a, c) 293T cells were transfected with expression vectors for the indicated HA-tagged VHHs and structural proteins of IAV (a) or VSV (c) fused to Renilla luciferase. Lysates of the respective cells were incubated with immobilized anti-HA antibodies in 96-well plates. Wells were washed and incubated with Renilla luciferase substrates to measure co-purified luciferase activity. Emitted light was normalized to luciferase activity in the lysate. Data from three independent experiments ± s.e.m. is displayed. (b,d) Purified IAV NP (b) or VSV N-RNA (d) was pre-incubated with the His₆-tagged VHHs indicated at the top and subsequently subjected to immunoprecipitation with the specified biotinylated VHH. Precipitation of NP/N and the respective VHH was analyzed by SDS-PAGE and colloidal Coomassie staining. Competition due to overlapping binding epitopes of the VHHs is indicated with red squares, successful co-purification with green squares. Representative data from at least three experiments is displayed.

Figure 5

Anti-IAV VHHs block nuclear import of vRNPs and mRNA transcription. (a, b) A549 cells or clones expressing the indicated VHHs were treated with 1 μg/mL Dox for 24 h and infected with IAV WSN (MOI 230) in the presence of 1 mM cycloheximide for 4 h. Controls were treated with 50 nM bafilomycin A1 (BafA). Cells were stained for NP, HA, DNA, and actin; Z-stacks were recorded by confocal microscopy and Z projections of representative examples are displayed in (a). Scale bars represent 20 μm. NP staining in the nucleus and cytoplasm was quantified with CellProfiler and ratios of nuclear/cytoplasmic signal intensities were quantified and normalized to untreated cells (nuclear import = 1.0) and BafA-treated cells (nuclear import = 0). Values from three independent experiments ± s.e.m. are shown. (c, d) 293T cells were transfected with expression vectors for IAV WSN PA, PB1, PB2, NP, pPolI-EGFP, and the indicated HA-tagged VHHs. 24 h post transfection, cells were stained for HA and analyzed by flow cytometry. The fraction of VHH-HA-positive cells that expressed high levels of EGFP was quantified. Exemplary histograms are shown in (c), and average data from three independent experiments ± s.e.m. are displayed in (d).

Figure 6

Anti-VSV VHHs impair mRNA transcription. (a,b) A549 cells or clones expressing the indicated VHHs were treated with 1 μg/mL Dox for 24 h, infected with VSV Indiana, and viral RNAs metabolically labeled with [³H]-uridine. We purified the RNA from cell lysates and separated RNA species by acid agarose-urea gel electrophoresis. An autoradiogram representative of three independent experiments is shown; the positions of the mRNAs of the respective viral genes are indicated. Band intensities of G, N, and P/M mRNAs were quantified and normalized to band intensities in the absence of Dox. Average values from three independent experiments ± s.e.m. are shown. (c) To test the effects of anti-VSV VHHs on polymerase activity in vitro, we incubated recombinantly expressed L and P with N-RNA templates purified form VSV virions. Reactions were performed in the absence or presence of the indicated VHHs as well as NTPs, including [α-³²P]-GTP. RNA species were separated by acid agarose-urea gel electrophoresis. An autoradiogram representative of three independent experiments is shown; the positions of the mRNAs of the respective viral genes are indicated.