Dissection of early molecular signalling during porcine reproductive and respiratory syndrome viral infection in macrophages

More than 20 years after its emergence, the porcine reproductive and respiratory syndrome (PRRS) is still having major impacts on pig health and welfare. PRRS results in reproductive failure and respiratory disorders. The etiologic agent is the PRRS virus which enters alveolar macrophages via a mechanism of receptor-mediated endocytosis which involves heparan sulphate, the macrophage marker sialoadhesin (PoSn) and CD163, a macrophage haemoglobin scavenger receptor. The virus is able to establish a persistent infection in pigs through suppression of the host immune response and elicits a high rate of mutation during viral replication. Since the early 1990s, vaccine therapy and management practices strategies have had a limited impact on the spread of the disease. PRRS remains a challenge to the sustainability of pig production, especially with the emergence of new highly pathogenic PRRSV strains. To understand how PRRSV activates, disturbs and exploits the host's immune response for its own benefit we propose this postgraduate studentship research project to dissect the early molecular signalling during PRRSV macrophage infection. The project will build upon the results from analyses of changes in host gene expression in macrophages infected in vitro with PRRSV and the discovery by the Pfizer industrial partner of CD163 as a key PRRSV co-receptor. The earlier BBSRC-funded project conducted at The Roslin Institute on host macrophage transcriptional responses to infection with PRRSV revealed several hundred genes whose expression was altered following infection. A rapid and intense host transcriptional remodelling was observed during the early phase of the replication of the virus which correlated with transient repression of type-I interferon transcripts as early as 8h post-infection. There was also evidence of differences in response due to host genotype. From the analysis of the early PRRS-regulated transcripts dataset a major class of regulatory proteins involved in reversible phosphorylation (protein kinases and phosphatases) was detected mostly during the first few hours post infection. Reversible phosphorylation of proteins is a major mechanism for the control of intracellular signalling cascades and maintenance of cellular response. The proposed CASE postgraduate studentship research project will focus on the role of host protein kinases and phosphatases whose expression is altered following infection and identify new PRRSV-regulated CD163-interactors in infected macrophages. The overall aim is to dissect the early molecular events associated with PRRSV infection in macrophages. Two initial lines of research will be pursued: 1. To determine the role of PRRSV-regulated protein kinases and phosphatases transcripts during early infection. The pattern of expression of transcripts encoding protein kinases and phosphatases will be reassessed using real-time PCR with RNA isolated from Pietrain and Landrace macrophages infected with European and American PRRSV isolates and a vaccine strain. The effects of loss/gain of function of selected protein kinases and phosphatases transcript on PRRSV infection will be examined. The effects of specific protein kinases and phosphatases inhibitors on PRRSV growth will be examined. 2. To identify CD163-protein interactors in productive (Pietrain) and non-productive (Landrace) PRRSV infections. Prior to the arrival of the student a yeast-2-hybrid library will be commissioned using RNA from PRRSV infected/non-infected Pietrain and Landrace macrophages and screened with CD163. Candidate CD163-interactors will be assessed using in-vitro pull down experiments. The function of the newly identified CD163-interactors will be investigated by loss-and/or gain-of-function approaches and localisation in PRRSV infected macrophages. The effects of the loss/gain of function of the identified CD163-interactors on PRRSV infection will be examined.