Nuclear localization of avian polyomavirus structural protein VP1 is a prerequisite for the formation of virus-like particles

Abstract

Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.

FIG. 1.

Schematic presentation of the method used for the generation of recombinant influenza viruses. The transfer plasmid carrying the gene of interest is cotransfected into 293T cells, together with booster plasmids expressing influenza virus PA, PB1, PB2, and NP genes. After 24 h, during which the recombinant vRNA has been transcribed from the transfer plasmid, cells are infected with influenza helper virus for packaging of the vRNA. At 24 h after infection, the supernatant containing the recombinant influenza viruses is used for the infection of CE cells in which the gene of interest is expressed.

FIG. 2.

Demonstration of APV late gene products in CE cells infected with recombinant influenza viruses expressing VP1, VP2, VP3, VP4, or VP4Δ. At 24 h after infection, the cells were analyzed by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, followed by immunoblotting using polyclonal antibody directed against APV particles. M, molecular mass markers, indicated on the left; APV, CE cells 4 days after infection with APV. The positions of the APV structural proteins are indicated. As gels with 12.5% acrylamide were used, VP3 and VP4 were not distinctly separated.

FIG. 3.

Subcellular localization of VP1 and VP4 in CE cells, demonstrated by indirect immunofluorescence using a monoclonal antibody directed against VP1 (left) or a polyclonal antibody directed against VP4 (right). Top row, analysis 8 h after infection with VP1 (VP1 influenza)- or VP4 (VP4 influenza)-expressing influenza viruses; middle row, analysis 2 days after transfection with plasmids expressing VP1 (VP1 transfection) or VP4 (VP4 transfection); bottom row, analysis 3 days after infection with APV (APV infection).

FIG. 4.

Subcellular localization of VP1 and VP4 after expression of VP1 (lane 1) or VP4 (lane 2) or after coexpression of different combinations of VP1, VP3, and VP4 (lanes 3 to 6) in CE cells. The cells were analyzed by indirect immunofluorescence 8 h after (co)infection with VP1-, VP3-, or VP4-expressing influenza virus using a monoclonal antibody directed against VP1 (VP1-specific staining; upper row) or a rabbit serum directed against VP4 (VP4-specific staining; lower row).

FIG. 5.

Demonstration of the colocalization of VP1 and VP4 using double-labeling immunofluorescence of CE cells coinfected with recombinant influenza viruses. Immunofluorescence was performed 8 h after infection using the monoclonal antibody 3G10G5 and anti-mouse-FITC conjugate for VP1-specific staining and the polyclonal antibody α1a directed against VP4 in combination with anti-rabbit-TRITC conjugate (VP4-specific staining). Upper row, coinfection with VP1- and VP4-expressing influenza viruses; lower row, coinfection with VP1-, VP3-, and VP4-expressing influenza viruses.

FIG. 6.

Analysis of APV VLP formation by density gradient centrifugation, immunoblotting, and electron microscopy. CE cells were sonicated 24 h after infection with different combinations of VP1-, VP3-, and VP4-expressing influenza viruses. (A) The presence of the proteins in the lysate was demonstrated by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis, followed by immunoblotting using a polyclonal antibody directed against APV particles. (B) Lysates were subjected to Nycodenz density gradient centrifugation, and fractions with buoyant densities corresponding to that of APV particles were collected, subjected to centrifugation, and subsequently analyzed by immunoblotting as described above. The positions of the APV structural proteins are indicated. M, molecular mass markers (from top to bottom: 97.4, 67.2, 45.0, 31.0, and 21.5 kDa). (C and D) Electron microscopic demonstration (uranyl acetate staining) of VLP purified by Nycodenz density gradient centrifugation from CE cell lysates coinfected with VP1- and VP3-expressing influenza viruses (C) or with VP1-, VP3-, and VP4-expressing influenza viruses (D). The inset in panel C shows a full particle. Bars, 100 nm.

FIG. 7.

Alignment of N-terminal VP1 amino acid sequences of 10 polyomaviruses. The abbreviations of the designations of polyomaviruses are according to van Regenmortel et al. (41). GHPV, goose hemorrhagic polyomavirus according to Johne and Müller (13). Basic amino acids potentially serving as an NLS are in boldface.

FIG. 8.

Analysis of the subcellular localization of VP1-NLS^SV40 and VLP formation. (A) Immunofluorescent staining of CE cells 8 h after infection with VP1- or VP1-NLS^SV40-expressing influenza viruses using a monoclonal antibody against VP1. The amino acid sequences of the N termini of both proteins are indicated (exchanged residues are italicized). (B) (Left) Electron microscopic demonstration (uranyl acetate staining) of VLP purified from CE cell lysates infected with VP1-NLS^SV40-expressing influenza viruses and concentrated by CsCl density gradient centrifugation. (Right) APV particles. Different types of particles, either empty (solid arrows) or full (open arrows), as well as a smaller particle (shaded arrow), are indicated. Bars, 100 nm.