Prospects for development of African swine fever virus vaccines.

African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naïve pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.

A viral mechanism for inhibition of the cellular phosphatase calcineurin.

The transcription factor NFAT (nuclear factor of activated T cells) controls the expression of many immunomodulatory proteins. African swine fever virus inhibits proinflammatory cytokine expression in infected macrophages, and a viral protein A238L was found to display the activity of the immunosuppressive drug cyclosporin A by inhibiting NFAT-regulated gene transcription in vivo. This it does by binding the catalytic subunit of calcineurin and inhibiting calcineurin phosphatase activity.

African swine fever virus protein A238L interacts with the cellular phosphatase calcineurin via a binding domain similar to that of NFAT.

The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-kappaB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.

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

cDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleave this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs 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 P1-2A 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.

Myristoylation of foot-and-mouth disease virus capsid protein precursors is independent of other viral proteins and occurs in both mammalian and insect cells.

The myristoylation of the foot-and-mouth disease virus (FMDV) capsid precursor P1-2A and its amino-terminal cleavage product 1AB, expressed from subgenomic cDNA, has been analysed. The modification reaction is independent of other FMDV proteins and occurs in both mammalian and insect cells. Blocking of the myristoylation site does not prevent efficient processing of the FMDV capsid precursor. A cDNA cassette in which the leader protease sequence is substituted by an ATG codon produces myristoylated 1AB, indicating correct removal of the novel N-terminal methionine residue.

Intracellular expression and processing of foot-and-mouth disease virus capsid precursors using vaccinia virus vectors: influence of the L protease.

cDNA cassettes of FMDV have been constructed which encode the capsid precursor (P1-2A) alone or with the proteases L and 3C which are required for processing of this precursor to the products 1AB, 1C, and 1D. These cassettes have been analyzed using in vitro transcription and translation reactions and within cells using recombinant vaccinia viruses. Processing of the precursors occurred more efficiently in cells than in cell-free systems but similar properties were observed. It was not possible to isolate recombinant vaccinia viruses containing FMDV cassettes which included the intact coding sequence for the L protein. Deletion of part of the L sequence, which abolished its proteolytic activity, also abolished this incompatibility with vaccinia virus. The vaccinia recombinant, vTF7-3, which expresses the bacteriophage T7 RNA polymerase was used in transient expression studies using plasmids containing a T7 promoter upstream of the FMDV cassettes. Under these conditions it was possible to coexpress L, P1-2A, and 3C in the vaccinia-infected cells; each of the proteolytic activities was observed and correctly processed 1AB, 1C, and 1D were produced.

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.

Nuclear and nucleolar localization of an African swine fever virus protein, I14L, that is similar to the herpes simplex virus-encoded virulence factor ICP34.5.

PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.