Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles

Abstract

How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

FIG 1

Detection of viral proteins in cell lysates and supernatants. Western blots of lysates and concentrated supernatant of 293FT cells transfected with different plasmids. (A) Transfection of empty plasmid, pI.18/ANDV-GPC, or cotransfection of pI.18/ANDV-GPC and pCMV-Bios/ANDV-N. (B) Transfection of empty plasmid or pWRG/PUU-M(s2). ANDV Gn and N proteins were detected with anti-Gn 6B9/F5 and anti-N 7B3/F7, respectively. ANDV and PUUV Gc was detected with MAb anti-Gc 2H4/F6. No MAb against PUUV Gn was available.

FIG 2

Characterization of hantavirus VLPs. Negative-stain EM of concentrated supernatants from cells transfected with pI.18/ANDV-GPC (A to D) or pWRG/PUU-M(s2) (E to H). Negative-stain EM using phosphotungstic acid of unfixed ANDV VLPs (B), PUUV VLPs (E, F), glutaraldehyde-fixed ANDV VLPs (A, C), and PUUV VLPs (G). Immunogold EM of ANDV VLPs (D) and PUUV VLPs (H) stained with uranyl acetate using patient sera (1:100) and protein A conjugated to gold beads (20 nm). (I) Sucrose gradient sedimentation of ANDV VLPs and detection of VLPs through Western blotting using anti-ANDV Gc MAb 2H4/F6. Buoyant density was determined by refractometry, and relative intensity of Gc bands was quantified as arbitrary units using ImageJ (40). (J) Dynamic light scattering of ANDV VLPs at pH 7.4 and ANDV VLPs treated for 30 min with SDS 0.1%. Bars, 100 nm.

FIG 3

Antigenicity of hantavirus VLPs. ELISA plates were activated with concentrated ANDV or PUUV VLPs, and their reactivity was tested with sera derived from patients infected with different hantavirus species (ANDV, PUUV, Sin Nombre virus [SNV]). A Student t test was used for statistical evaluation: ***, P < 0.00025; **, P < 0.0025; *, P < 0.025.

FIG 4

Deletion analysis of ANDV Gc endodomain mutant. (A) Western blot analysis using anti-Gc and anti-actin MAbs of different fractions corresponding to the nonbiotinylated fraction (intracellular proteins), the biotinylated fraction (surface proteins), or the concentrated supernatant of 293FT cells that were transfected with pI.18/ANDV-GPC, pI.18/ANDV-GPCΔGcCT, or empty vector and biotinylated 48 h posttransfection. (B) Negative-stain EM analysis of concentrated supernatants derived from 293FT cells transfected with pI.18/ANDV-GPCΔGcCT. Bar, 100 nm.