Charles C. Abrams

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.

African Swine Fever Virus Protein A238L Interacts with the Cellular Phosphatase Calcineurin via a Binding Domain Similar to That of NFAT

ABSTRACT 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-κB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.

The MyD116 African Swine Fever Virus Homologue Interacts with the Catalytic Subunit of Protein Phosphatase 1 and Activates Its Phosphatase Activity

ABSTRACT The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-α catalytic subunit expressed as a histidine fusion protein (6×His-PP1α) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α). ICP34.5 recruits PP1 to dephosphorylate the α subunit of eukaryotic translational initiation factor 2 (also known as eIF-2α); possibly the ASFV DP71L protein has a similar function.

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.

Macrophage Transcriptional Responses following In Vitro Infection with a Highly Virulent African Swine Fever Virus Isolate

ABSTRACT We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1β and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells.

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.

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.

Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus.

African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus.

Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response

African swine fever virus (ASFV) encodes multiple copies of MGF360 and MGF530/505 gene families. These genes have been implicated in the modulation of the type I interferon (IFN) response. We investigated the effect of modulating the IFN response on virus attenuation and induction of protective immunity by deleting genes MGF360 (MGF360-10L, 11L, 12L, 13L, 14L) and MGF530/505 (MGF530/505-1R, 2R and 3R) and interrupting genes (MGF360-9L and MGF530/505-4R) in the genome of the virulent ASFV isolate Benin 97/1. Replication of this deletion mutant, BeninΔMGF, in porcine macrophages in vitro was similar to that of the parental virulent virus Benin 97/1 and the natural attenuated isolate OURT88/3, which has a similar deletion of MGF360 and 530/505 genes. Levels of IFN-β mRNA in macrophages infected with virulent Benin 97/1 isolate were barely detectable but high levels were detected in macrophages infected with OURT88/3 and intermediate levels in macrophages infected with BeninΔMGF. The data confirms that these MGF360 and MGF530/505 genes have roles in suppressing induction of type I IFN. Immunisation and boost of pigs with BeninΔMGF showed that the virus was attenuated and all pigs (5/5) were protected against challenge with a lethal dose of virulent Benin 97/1. A short transient fever was observed at day 5 or 6 post-immunisation but no other clinical signs. Following immunisation and boost with the OURT88/3 isolate 3 of 4 pigs were protected against challenge. Differences were observed in the cellular and antibody responses in pigs immunised with BeninΔMGF compared to OURT88/3. Deletion of IFN modulators is a promising route for construction of rationally attenuated ASFV candidate vaccine strains.

Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system

A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus VΔMGF-GUS. Subsequently the GUS gene was removed by site-specific recombination between the two loxP sites involving expression of the bacteriophage Cre recombinase enzyme to create recombinant virus VΔMGFΔGUS. The EP402R and EP153R genes were subsequently deleted from the genome of VΔMGFΔGUS, using the same GUS marker gene, to construct virus VΔMGFΔCD2-Lectin-GUS. These sequential deletions of ASFV genes were shown not to alter virus replication significantly.