Role of the C-terminal region of vervet monkey polyomavirus 1 VP1 in virion formation

Abstract

Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (ΔC VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and ΔC VP1 VLPs were similar in size, but the number of ΔC VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation.

Fig. 1.

Alignment of PyV VP1s in C-terminal portions. Alignment of PyV VP1s in C-terminal portions with that of VmPyV. The VP1 sequences of PyVs were obtained from GenBank (abbreviations and accession numbers are indicated in Table 1). Amino acid identities are shaded as follows: black shading indicates that all amino acid sequences were conserved, whereas grey shading indicates that more than 51% of them were conserved.

Fig. 2.

Expression of VmPyV VP1 protein in HEK293T cells. Immunocytochemical and immunoblot analyses (IC and IB) of VmPyV VP1 with the anti-SV40 VP1 antibody. The HEK293T cells were transfected with WT VP1, ΔC VP1 or the corresponding empty vector (Mock) as a negative control. (A) VmPyV VP1 was detected colored in green. Cell nuclei were stained with DAPI (blue color). Scale bar, 10 nm. (B) WT and ΔC VP1 signals were detected in cellular lysates from HEK293T cells at the expected molecular weight positions in immunoblotting. Actin was used as a loading control.

Fig. 3.

Electron micrographs of WT VP1 and ΔC VP1-expressing cells. Electron micrographs of HEK293T cells expressing WT VP1 (A–C) and ΔC VP1 (D–F). (B, C, E and F) Higher magnification of the regions indicated in panels A, B, D and E, respectively. All WT and ΔC VLPs were observed exclusively in the nuclei. The arrows indicate VLPs. N: nucleus.

Fig. 4.

Sucrose gradient sedimentation analyses. (A–C) Immunoblot analyses of VP1 in fractionated samples after sucrose gradient sedimentation of JCV VLPs and cellular lysates from HEK293T cells expressing WT VP1 or ΔC VP1. Cellular lysates were separated by 30–50% sucrose gradient sedimentation and fractionated into 12 fractions from the tops of the tubes. The 12 fractions were separated by SDS-PAGE and subjected to immunoblotting with the anti-SV40 VP1 antibody. (A) Purified JCV VLPs expressed in E. coli. (B) HEK293T cell lysates transfected with WT VP1 and (C) ΔC VP1. (D) Electron micrograph of negative staining of fractions 6 to 9 from HEK293T cells transfected with WT VP1. The arrows indicate VmPyV VLPs