Establishment of a CPER reverse genetics system for Powassan virus defines attenuating NS1 glycosylation sites and an infectious NS1-GFP11 reporter virus

Abstract

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. NS1 proteins are highly conserved glycoproteins that regulate replication, spread, and neurovirulence. POWV NS1 contains three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, and N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated, with 1-2 log decreases in titer. Severely attenuated recLI9-N224Q exhibited a 2- to 3-day delay in focal cell-to-cell spread and reduced NS1 secretion but was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations reduce POWV neuroinvasion in vivo. To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1-lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly, and secretion and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo and enables the rational genetic design of live attenuated POWV vaccines. IMPORTANCE Our findings newly establish a mechanism for genetically modifying Powassan viruses (POWVs), systematically defining pathogenic determinants and rationally designing live attenuated POWV vaccines. This initial study demonstrates that mutating POWV NS1 glycosylation sites attenuates POWV spread and neurovirulence in vitro and in vivo. Our findings validate a robust circular polymerase extension reaction approach as a mechanism for developing, and evaluating, attenuated genetically modified POWVs. We further designed an infectious GFP-tagged reporter POWV that permits us to monitor secretory trafficking of POWV in live cells, which can be applied to screen potential POWV replication inhibitors. This robust system for modifying POWVs provides the ability to define attenuating POWV mutations and create genetically attenuated recPOWV vaccines.

Keywords: NS1 glycosylation mutants; Powassan virus; attenuation; cell-to-cell spread; circular polymerase extension reaction; flavivirus; reverse genetics; split-GFP.

Fig 1

CPER generation and cell-to-cell spread of recombinant POWV strain LI9. (A) VeroE6 cells were infected with LI9 POWV at an MOI of 0.01 with or without anti-POWV HMAF (1:250) during adsorption. Cells were washed with PBS, and media were replaced with Dulbecco modified Eagle’s medium (DMEM) with or without anti-POWV HMAF (1:250) or control ascitic fluid 6 h post adsorption. Cells were methanol fixed 3 dpi and immunostained with anti-POWV HMAF (1:5,000) (20, 79, 80). (B) Schematic of the LI9 POWV genome and overlapping fragments amplified from LI9 cDNA. (C) CPER assembly schematic of F1-F5 fragments with a UTR-Linker fragment containing the last 26 nucleotides of the LI9 3′UTR, hepatitis delta virus ribozyme (HDVr), SV40 polyadenylation signal, a cytomegalovirus (CMV) promoter, and 33 nucleotides of the LI9 5′UTR. (D) Agarose gel electrophoresis of PCR-amplified fragments (F1–F5) showing a representative image of three experimental repeats. F1–F5 were combined in equal molar amounts with the UTR-Linker in a CPER. A representative agarose gel of CPER product. Resultant LI9 CPER products were transfected into HEK293T or VeroE6 cells, and supernatants were subsequently used to infect VeroE6 cells and rescue infectious recLI9 viruses. (E) Immunostaining CPER-transfected VeroE6 or HEK293T cells (7 or 3 dpt, respectively) display focal cell-to-cell spread foci morphologies. (F) Infectious recLI9 virus rescued from CPER transfected cells and grown in HEK293T cells vs CPER controls amplified without the UTR-linker fragment. (G) Comparison of WT LI9 and recLI9 focal cell-to-cell spread phenotypes in immunostained VeroE6 cells. (H) Growth kinetics of WT LI9 (red) and recLI9 (blue) POWVs 1–3 dpi in VeroE6 cells (MOI 1). Analysis repeated at >3 times.

Fig 2

CPER POWV NS1 glycosylation mutants have impaired replication kinetics. (A) Schematic of N-linked glycosylation sites within POWV NS1 proteins. (B) Alphafold2 model of dimeric POWV NS1 protein showing pink or blue monomers and the localization of putative N-linked glycosylation sites (N85, N208, N224) in red (84). (C) Schematic strategy for CPER-generating N-linked glycosylation site (NxT) mutants using overlapping fragments to introduce N to Q codon changes. Fragment 2 was split into subfragments 2A and 2B with overlapping regions incorporating mutations. (D) VeroE6 cells were infected with WT LI9, recLI9-NS1_N85Q, recLI9-NS1_N208Q, and recLI9-NS1_N224Q mutant viruses (MOI 1), and 7 dpi cell lysates and supernatants were analyzed by Western blot using antibodies POWV-NS1 (mAb), anti-POWV HMAF, or GAPDH. (E) Ratios of intracellular vs extracellular NS1 levels from representative Western blot (2D) of rec-LI9-NS1 glycosylation mutants were performed >2 times. (F) Growth kinetics of WT LI9 (black), CPER-generated NS1 glycosylation mutants recLI9-NS1_N85Q (purple), recLI9-NS1_N208Q (green), and recLI9-NS1_N224Q (pink) 1–6 dpi of VeroE6 cells (MOI 0.1). *Statistically significant by two-way ANOVA with Tukey’s post-test (P < 0.05). (G) Kinetic comparison of focal cell-to-cell spread by WT LI9 and recLI9-NS1 glycosylation mutants 1–6 dpi in VeroE6 cells (MOI 0.1) by anti-POWV (HMAF) immunoperoxidase staining of POWV-infected cells.

Fig 3

POWV NS1 mutant dimerization and glycosidase analysis. (A) VeroE6 cells were infected with WT POWV LI9, recLI9-NS1_N85Q, recLI9-NS1_N208Q, or recLI9-NS1_N224Q mutants (MOI, 1). Cell lysates were harvested 7 dpi with or without heat-directed dimer disassembly prior to Western blot analysis using anti-POWV-NS1 mAb (1:5,000) or anti-GAPDH (1:5,000). (B) VeroE6 cells were infected with WT LI9, recLI9-NS1_N85Q, recLI9-NS1_N208Q, or recLI9-NS1_N224Q mutant viruses (MOI, 1). Cell lysates were harvested 7 dpi, and 20 µg of protein lysate or 50 µL of supernatant was subjected to Endo H or PNGase F digestion. Undigested and digested samples from cell lysate or supernatant were analyzed by Western blot using antibody to POWV-NS1 or GAPDH.

Fig 4

POWV N224Q mutation attenuates neurovirulence in C57BL/6 mice. (A) Body weight analysis and (B) Kaplan-Meier survival curves (LI9 vs recLI9-N224Q, P = 0.0442) of pups of C57BL/6 mice (N = WT:5; N224Q:5; Cont:3) intracranially inoculated with 2 × 10² FFU of WT LI9, recLI9-N224Q, or buffer only. (C) Body weight analysis and (D) Kaplan-Meier survival curves (LI9 vs N224Q, P = 0.3310, not significant) for C57BL/6 mice (male, N = WT:8; N224Q:10; Cont:4) footpad inoculated with 2 × 10³ FFU of WT LI9, recLI9-N224Q, or a buffer-only control.

Fig 5

Generation of a split GFP POWV reporter virus. (A) Schematic of the split GFP fluorescence reporter system directed by the recLI9-NS1-GFP11 virus. Individual NS1 monomers (blue and pink) with a 16 residue GFP11 tag added to the NS1 C-terminus (yellow) with co-expressed, nonfluorescent, GFP_1–10 (gray). The recLI9-NS1-GFP11 expression of NS1-GFP11 protein reconstitutes GFP fluorescence in cells co-expressing GFP_1–10. (B) CPER strategy for POWV-NS1-split11 generation. F2 was split into subfragments F2A and F2B with primer-directed incorporation of GFP11 sequences. (C) Retrovirus-transduced HEK293T cells constitutively expressing mCherry (cytoplasm) and either ER-localized ER-GFP_1–10 (mCh-ER-GFP_1–10) or cytoplasm-localized GFP_1–10 (mCh-GFP_1–10) were infected with recLI9-NS1-GFP11. Live imaging captured 2–3 dpi shows foci of GFP fluorescence in LI9-NS1-GFP11-infected cells expressing ER translocated GFP_1–10 but not cytoplasmically expressed GFP_1–10. (D) Growth kinetics of WT LI9 (red) and recLI9-NS1-GFP11 (blue) 1–3 dpi in VeroE6 cells (MOI 1). (E) Immunostaining of LI9-NS1-GFP11 infected VeroE6 cell foci 3 dpi with anti-POWV HMAF. (F and G) Retrovirus-transduced VeroE6 cells expressing mCherry-ER-GFP_1–10 were LI9-NS1-GFP11 infected, and 6 dpi cells paraformaldehyde fixed. Representative images show perinuclear NS1-GFP fluorescence (F) and the discrete accumulation of NS1-GFP in large intracellular vesicles 6 dpi (G). Bars represent 10 µm.