Mechanism of action of an African swine fever virus virulence factor

Lead Research Organisation: St George's University of London
Department Name: Basic Medical Sciences

Abstract

African swine fever virus (ASFV) causes an economically important disease of pigs with high mortality rates. We plan to study one of the proteins (DP71L) encoded by the virus to determine its function in cells and the role it plays in causing disease in pigs. Previous work has shown that deleting the gene for this protein can reduce ASFV virulence in pigs. This protein is similar over part of its length to a Herpes simplex virus virulence factor, ICP34.5, and to a host protein (GADD34) which is induced by factors such as damage to DNA. We have recently shown that DP71L has two novel functions not demonstrated for ICP34.5 or GADD34. First it interferes with induction of interferon (IFN), which activates an important host anti-viral pathway. The mechanism by which it does this is not known. Secondly, DP71L stimulates a cellular enzyme, protein phosphatase 1 (PP1), also by an unknown mechanism. PP1 can be directed to activate or inhibit other cellular pathways by association with regulatory proteins. We propose that the DP71L protein acts as a regulator of PP1 to displace an inhibitor from PP1 and target the enzyme to modulate the activity of host cell pathways. This may be the mechanism by which DP71L inhibits induction of IFN. Our prediction is that DP71L may inhibit other cellular functions. Mutant forms of the DP71L protein will be made and used to study which parts of the protein are needed for each of its functions. We will also determine at which point DP71L blocks the IFN induction pathway and investigate if DP71L affects other host pathways. In particular we will study whether DP71L affects expression of other cellular genes, in addition to IFN, which are important for stimulating the host's response to infection. We will also study whether DP71L blocks two other host pathways which restrict virus replication. In these pathways the host cell either commits suicide (called apoptosis) or shuts-off translation of messenger RNAs to prevent virus from replicating. Deletion of the DP71L gene dramatically reduces virulence in pigs. By making recombinant virus which expresses mutant forms of the DP71L protein, we will determine if either or both functions of the protein are required for virulence. We will also determine if the functions we observe in isolated cells are also observed in infected pigs. The results will help us to understand more about the mechanisms by which IFN is induced, and the functions of PP1 and how this may be directed by viruses to avoid the host's defences. Understanding the role of individual proteins in virulence will assist in the rational development of an attenuated ASFV vaccine. Currently no vaccine is available so disease control relies on rapid diagnosis and implementation of quarantine and slaughter.

Technical Summary

We will investigate the function of an African swine fever virus (ASFV)-encoded protein, DP71L which we have recently shown both inhibits activation of transcription from the interferon (IFN)-beta promoter and stimulates protein phosphatase 1 (PP1) activity in infected cells. Our data has shown that DP71L can inhibit both IRF-3- and NF-kappaB-dependent gene transcription, suggesting it may have a broader effect on host immunomodulatory gene transcription. The DP71L protein is expressed as either a long form localised mainly in the nucleolus or a short form localised mainly in the nucleus of cells. Deletion of the short form of the gene dramatically reduces virulence in pigs. We propose that DP71L acts as a regulatory subunit of PP1 to activate and target the enzyme to dephosphorylate specific substrates in the nucleus and suggest that this is the mechanism by which IFN induction is inhibited. We will map the domains in DP71L that are required to bind to and stimulate PP1 and to inhibit IFN. This will establish if these two activities are linked. We will also determine at which point DP71L blocks IFN induction and establish if this is linked to inhibition of NF-kappaB activation. We will also investigate if DP71L modulates transcription of other immunomodulatory genes. To determine if these activities of DP71L are linked to its role in virulence, we will replace the wild-type gene on the virus genome with mutant DP71L genes which do not have these functions. Virulence will be studied in pigs. We will also monitor the in vivo effect of DP71L expression on induction of IFN and activation of IFN-induced pathways as well as other predicted functions of DP71L. The results will improve our understanding of the IFN induction pathway as well as revealing novel functions for PP1 and how these may be targeted by viruses. In addition we will learn more about mechanisms of virus pathogenesis which will help in the rational design of attenuated virus vaccines. Joint with [BB/E021239]
 
Description 1. The characterisation of the molecular basis of how the DP71L(s) gene product of African Swine Fever Virus (ASFV) targets the host translational initiation factor eIF2alpha for dephosphoryation, thus preventing the host from switching off translation in response to dsRNA and ER stress (published in Zhang et al., 2010; J. Virol. 84, 10681-9) 2. The characterisation of mutant forms of DP71L(s) that affect IFN induction. A form that is targeted to the nucleus still limits IFN induction despite being unable to down-regulate eIF2alpha phosphorylation, implying that the mechanism for inhibiting IFN induction is distinct from the block to translational shut-off. A form that is unable to recruit the catalytic subunit of protein phosphatase 1 shows only a limited ability to block IFN induction implying that the mechanism of efficient inhibition of IFN induction requires phosphatase recruitment.
Exploitation Route We have identified DP71L as a potent inhibitor of stress-mediated translational arrest and other stress responses. Since DP71L is a small protein we anticipate that it may be a useful reagent in scaled-up recombinant protein synthesis applications, and the IAH/Pirbright technology transfer office has funded a small scale project to look at this. We have also used our data as the basis of a further PhD student project based in Linda Dixon's lab at Pirbright (current as of November 2014)
Sectors Agriculture, Food and Drink

 
Description Our studies on DP71L have generated a spin-off project funded by IAH technology transfer program.