Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system

Abstract

cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleaves this molecule to lAB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant Ws constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P 1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.

Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

“…This has allowed us to investigate the inherent properties of both the natural and mutated forms of the capsid precursor protein with respect to antigenicity, receptor binding, and self-assembly into higher-order complexes. The capsid precursor proteins of most picornaviruses (except, for example, hepato-and parechoviruses) are modified by the covalent addition of a myristate group at the N terminus, and there is evidence that this plays a role in their assembly into pentameric viral subunits (1,36,37,43). The C termini of the capsid precursor proteins of different genera of the picornaviruses differ with respect to the presence or absence of the nonstructural protein, 2A.…”

“…These three components remain associated in a protein complex which now acts as a monomer for the self-assembly of five monomers into the pentameric capsid subunit. The assembly of higher-order structures from monomeric subunits has been shown for several picornaviruses to be dependent on N-terminal myristoylation of the precursor (1,36,37,43). The myristoyl moieties in the mature PV are clustered near the fivefold vertices on the capsid interior and participate in stabilizing interactions between the five protomer subunits that form the pentamer (13,27).…”

“…Earlier studies demonstrated the production of empty capsids of several picornaviruses following translation of the viral RNA in rabbit reticulocyte lysates (25,30,45) or expression of capsid precursor in bacterial (34), yeast (55), insect (14) and mammalian (1,3) cells. In these studies, capsid precursors were coexpressed with the viral protease in order for capsid assembly to occur within the expression system.…”

Evidence of partial protection against foot-and-mouth disease in cattle immunized with a recombinant adenovirus vector expressing the precursor polypeptide (P1) of foot-and-mouth disease virus capsid proteins.

“…For this study we cloned into an adenovirus vector the gene which expresses the precursor polyprotein of the four capsid proteins (P1) of FMDV. This polyprotein was chosen as it has been shown to contain most of the continuous and discontinuous B cell epitopes involved in the induction of neutralizing antibodies to FMDV (Bittle et al, 1982 ;Strohmaier et al, 1982 ;Saiz et al, 1994 ;Abrams et al, 1995 ;Brown, 1995 ;Mateu, 1995), as well as T-cell epitopes recognized by cattle and swine (Collen, 1994 ;Rodriguez et al, 1994 ;van Lierop et al, 1995). The conformation of the expressed products of this gene cloned into baculovirus and vaccinia vectors has been shown to be antigenically similar to native capsid proteins (Saiz et al, 1994 ;Sanz-Parra et al, 1998).…”

Influence of the Leader protein coding region of foot-and-mouth disease virus on virus replication

“…Initiation at the Lb start site should produce the authentic capsid precursor; however, initiation of translation at the Lab start site will result in an N-terminal extension to the P1-2A protein resulting in a product that cannot be modified by the cellular myristoylation system (Towler et al, 1988). Loss of myristoylation results in defective picornavirus capsid assembly or stability (Abrams et al, 1995;Belsham et al, 1991;Chow et al, 1987). Thus, the presence of the Lab start site, in addition to the Lb start site, could compromise the viability of the mutant FMD viruses lacking Lb alone.…”