Construction and Crystal Structure of Recombinant STNV Capsids

Abstract

A codon-optimised gene has been expressed in Escherichia coli to produce the coat protein (CP) of the Satellite Tobacco Necrosis Virus. This protein assembles in vivo into capsids closely resembling those of the T = 1 wild-type virus. These virus-like particles (VLPs) package the recombinant mRNA transcript and can be disassembled and reassembled using different buffer conditions. The X-ray crystal structure of the VLP has been solved and refined at 1.4 Å resolution and shown to be very similar to that of wild-type Satellite Tobacco Necrosis Virus, except that icosahedral symmetry constraints could be removed to reveal differences between subunits, presumably owing to crystal packing. An additional low-resolution X-ray crystal structure determination revealed well-ordered RNA fragments lodged near the inside surface of the capsid, close to basic clusters formed by the N-terminal helices that project into the interior of the particle. The RNA consists of multiple copies of a  3-bp helical stem, with a single unpaired base at the 3′ end, and probably consists of a number of short stem–loops where the loop region is disordered. The arrangement of the RNA is different from that observed in other satellite viruses.

Introduction

Satellite Tobacco Necrosis Virus (STNV) is one of the smallest known viruses. There are three serotypes, STNV-1, STNV-2 and STNV-C, that infect tobacco plants but only in the presence of its helper virus Tobacco Necrosis Virus. In vivo, it is transmitted by the soil fungus Olpidium brassicae to the root cells of the plant. STNV-1 has a  1239-nt single-stranded RNA genome, with the structural gene for the coat protein (CP) starting close to the 5′ terminus, the AUG initiation codon lying at position 30–32 of the RNA strand.¹ The protein consists of a 195-amino-acid polypeptide that lacks an N-terminal methionine. Sixty copies of the CP subunit are required to form the T = 1 capsid. STNV crystallises readily, and crystals have been observed in vivo,² leading to early X-ray analysis.³ These structural studies have concentrated on STNV-1, whose X-ray crystal structure was solved at 3 Å resolution⁴ and later refined to 2.5 Å.⁵ An expanded form, produced by ethylenediaminetetraacetic acid treatment, has also been solved at 2.8 Å resolution.⁶ The icosahedrally averaged electron density maps clearly show residues 12–195 of the CP, but the 11 N-terminal residues, which lie towards the centre of the particle, appear disordered. The interior of the particle showed no electron density corresponding to an ordered RNA structure in these studies. A neutron crystal structure at 16 Å resolution, using H₂O/D₂O contrast matching, shows good density that probably corresponds to RNA in a region just inside the protein capsid, but the resolution is too low to show any detail.⁷ The structure of the viral RNA and its interaction with the CP remain to be determined, and little is known about STNV assembly or whether specific packaging signals are present in the RNA genome. Like other small T = 1 satellite viruses whose structures are known at high resolution, such as Satellite Tobacco Mosaic Virus (STMV)8, 9 and Satellite Panicum Mosaic Virus (SPMV),10, 11 STNV is a useful model system to study RNA packaging and self-assembly of simple icosahedral capsids.

Virus-like particles (VLPs) are self-assembling particles based on virus components, which have found extensive use in synthetic vaccines.¹² In a number of cases, subviral particles that do not contain the viral genome and thus lack infectivity while retaining the antigenic properties necessary for successful vaccines can be prepared. In addition, VLPs are rapidly finding applications as scaffolds in nanoparticle biotechnology, as they can be very stable and allow chemical modification to attach specific molecules such as fluorescent labels or metal clusters.¹³ The T = 1 plant satellite viruses could provide a useful additional class of VLPs, especially given their small sizes. We sought, therefore, to produce a recombinant expression system for STNV in Escherichia coli. Previously, a truncated version of the CP of the T = 3 Sesbania Mosaic Virus was expressed in E. coli, where it assembled into T = 1 subviral particles whose crystal structure was determined.14, 15 Expression of the STNV CP in E. coli has been reported, but the product appeared proteolytically unstable, and no self-association into VLPs was observed.¹⁶

Here, we describe the generation of a synthetic gene for STNV CP, with optimised codon usage to allow expression in E. coli, thus allowing for extensive future mutagenesis. The recombinant material assembles spontaneously into stable T = 1 capsids that are readily purified and can be disassembled and reassembled using a set of defined buffer conditions. We have solved and refined the X-ray crystal structure of this VLP to 1.4 Å resolution, which, as expected, shows close similarity to that of the normal virus, and a low-resolution study reveals regions of double-helical RNA bound to the inner surface of the capsid.