A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7.

The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.

African swine fever virus protein p54 interacts with the microtubular motor complex through direct binding to light-chain dynein.

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.

Rinderpest virus C and V proteins interact with the major (L) component of the viral polymerase.

Rinderpest virus, like other Morbilliviruses, expresses three proteins from the single P gene. In addition to the P protein, which interacts both with the viral polymerase (L) and the nucleocapsid (N) protein, the virus expresses a C and a V protein from the same gene. The functions of these two proteins in the viral life cycle are not clear. Although both C and V proteins are dispensable, in that viable viruses can be made that express neither, each seems to play a role in optimum viral replication. We have used the yeast-two hybrid system, binding to coexpressed fusions of C and V to glutathione-S-transferase, and studies of the native size of these proteins to investigate interactions of the rinderpest virus C and V proteins with other virus-encoded proteins. The V protein was found to interact with both the N and L proteins, while the C protein was found to bind to the L protein, and to self-associate in high-molecular-weight aggregates.

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.

An ARID family protein binds to the African swine fever virus encoded ubiquitin conjugating enzyme, UBCv1.

The NH(2)-terminal end of a protein, named SMCp, which contains an ARID (A/T rich interaction domain) DNA binding domain and is similar to the mammalian SMCY/SMCX proteins and retinoblastoma binding protein 2, was shown to bind the African swine fever virus encoded ubiquitin conjugating enzyme (UBCv1) using the yeast two hybrid system and in in vitro binding assays. Antisera raised against the SMCp protein were used to show that the protein is present in the cell nucleus. Immunofluorescence showed that although UBCv1 is present in the nucleus in most cells, in some cells it is in the cytoplasm, suggesting that it shuttles between the nucleus and cytoplasm. The interaction and co-localisation of UBCv1 with SMCp suggest that SMCp may be a substrate in vivo for the enzyme.

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.

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.