The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles

Masa-Aki Kawano,Takamasa Inoue,Hiroko Tsukamoto,Tatsuya Takaya,Teruya Enomoto,Ryou-U Takahashi,Naoki Yokoyama,Noriaki Yamamoto,Akira Nakanishi,Takeshi Imai,Tadashi Wada,Kohsuke Kataoka,Hiroshi Handa

doi:10.1074/jbc.m511261200

Abstract

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.

Highlights

SV40 is a small, nonenveloped DNA tumor virus that belongs to the family Polyomaviridae
VP1 pentamers do not assemble into particles under physiological salt and pH conditions (150 mM NaCl, pH 7.0) (Fig. 1A, top panel)
Addition of HF-VP2 to VP1 pentamers resulted in particle formation under physiological salt and pH conditions (Fig. 1A, middle panel)

Summary

Introduction

SV40 is a small, nonenveloped DNA tumor virus that belongs to the family Polyomaviridae. We prepared highly purified SV40 VP1 pentamers [9, 25, 26], which assembled into morphologically heterogeneous particles under various nonphysiological conditions [26]. Under physiological salt and pH conditions, pentamers do not assemble into particles in vitro [26], suggesting that component(s) that promote proper assembly are lacking. We identified the functional domains of VP2 that promote VP1 assembly These results provide information on the mechanism of SV40 virus assembly and suggest a new approach to construct SV40 viral vectors in vitro

Objectives

Results

Conclusion