Understanding the CD163 - PRRS virus interaction to improve genetic engineering for resistance

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Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic viral disease of pigs that causes major economic losses. The causative agent of PRRS, PRRS virus (PRRSV) is a rapidly evolving small enveloped RNA virus. Whilst improvements have been effected with changes in husbandry and vaccination, PRRS still has major impacts on pig health and welfare. PRRS accounts for about a third of the cost of infectious disease to the US pig industry amounting to $650M per year. PRRS is the most costly disease to pig industries of Europe, China and North America and new PRRSV variants have the potential to be even more devastating as demonstrated by recent outbreaks of highly pathogenic variants of the virus in China and Southeast Asia. PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. Like all viruses PRRSV relies on the host cell machinery to replicate itself and generate new viral particles. One of these host proteins, CD163, has been found to be essential for the virus' escape from the uptake vesicle during the entry process by membrane fusion. CD163 is expressed on the surface of macrophages and the extracellular portion of CD163 forms a pearl-on-a-string structure of nine scavenger receptor cysteine-rich (SRCR) domains. SRCR domain 5 (SRCR5) was found to be essential for PRRSV's ability to infect a host cell. In addition to mediating PRRSV infection, CD163 has a variety of important functions within the host cell, such as removing haemoglobin-haptoglobin complexes from the blood, thereby preventing the formation of reactive oxygen species that could damage other cells.My recent research together with others demonstrated that genome editing is a viable tool to combat PRRSV infection and confirmed the importance of CD163 in in this process. Work by another research group has shown that pigs in which the CD163 gene was knocked-out are resistant to PRRSV infection. However, the expression and function of CD163 in these animals is completely abolished and thus their ability to remove haemoglobin/haptoglobin complexes from blood, ischaemic repair and inflammation response may be impaired. My work removed SRCR5 by deletion (deltaSRCR5) of the encoding exon from the pig genome. I found that macrophage cells from these animals were not only resistant to PRRSV infection but also expressed the truncated deltaSRCR5 CD163 on the macrophage surface whilst maintaining haemoglobin-haptoglobin scavenger function.It is expected that removal of SRCR5 and thereby destruction of the "lock" abrogates the key-lock interaction of PRRSV with its receptor. Thereby, it prevents fusion of PRRSV with the host cell membrane. However, we do not understand enough of the CD163-PRRSV interaction to exclude the potential for this highly mutagenic RNA virus to adapt to a deltaSRCR5 cell or pig. Therefore, we need further understanding of the CD163 - PRRSV interaction and insight into PRRSV's ability to adapt to the deltaSRCR5 modification. My research on deltaSRCR5 shows that removal of a single exon from CD163 can abolish PRRSV infection, emphasising the great power of genome editing technology to induce subtle genetic changes.The aim of this project is to further assess the CD163 - PRRSV interaction and the potential of the PRRSV to adapt to a deltaSRCR5 pig as well as to generate even more subtle changes to the pig genome than exist in the current deltaSRCR5 pigs. I propose the following strategies to address these questions: to identify domains and amino acids of CD163 interacting with PRRSV; to assess the PRRSV-CD163 signalling and interactome; to serially passage PRRSV on cells from deltaSRCR5 pigs to mimic virus evolution; to produce functional deltaSRCR5 pigs by exon skipping rather than exon deletion.