Construction of a Synthetic Viral ORFeome for Identifying Regulators of Host Immune Responses

Abstract

Advances in DNA synthesis and genetic screening technologies enable the systematic exploration of gene function. Previous screening technologies, such as large-scale ORFeome libraries and CRISPR-Cas9 libraries either relied on collections of ORFs cloned from the cell or the synthesis of short segments of DNA corresponding to target genes. Smaller scale ORF libraries from pathogens, such as Kaposi’s sarcoma herpesvirus and herpes simplex virus 1, were constructed by amplifying target DNA from the viral genome and subcloning it into an expression vector. To date, DNA synthesis technology has not been used to construct a largescale collection of ORFs from human or viral sources. We generated a large-scale collection of viral ORF products, the viral ORFeome, from over 600 strains of virus that were known to infect humans or closely related to viruses that infect humans and representing a potential zoonotic threat. The viral ORFeome contains over 10,000 unique viral ORF fragments with up to five unique barcodes each that can be individually expressed in mammalian cells for genetic perturbation screens, which we demonstrated by performing genetic screens for viral ORFs that regulate cellular proliferation. We then employed this viral ORFeome to identify viral ORFs that modulate the host immune response. First, we performed a viral ORFeome screen for viral ORFs that regulate the expression of class I MHC on the cell surface. Class I MHC expression is critical for CD8+ T cell mediated killing of virally infected cells. We identified several novel regulators of class I MHC cell surface expression, including MC162R. We demonstrated that MC162R engages the host cell ubiquitin machinery and redundantly recruits several host ubiquitin E3 ligases to facilitate the ubiquitylation and degradation of class I MHC. Second, we sought to examine whether the viral ORFeome could be used to examine viral inhibition of cell signaling pathways, focusing on the IFNb and IFNg signaling pathways. IFN signaling is critical for generating an antiviral state in response to viral infection and are frequently impaired by viral proteins. We identified several novel regulators of IFN signaling, including families of viral proteins that preferentially regulated either IFNb or IFNg signaling. We characterized one of these families of viral ORFs, the yatapoxvirus 151R ORFs, as selective inhibitors of IFNb signaling that interact with IRF9. Finally, we sought to develop a system for screening for regulators of nucleic acid sensing. We developed a knock-in reporter system for IFNB1 expression and leveraged the ability of Sendai virus to potently induce RNA sensing to conduct a screen for viral ORFs that regulate RNA sensing. Collectively, these results demonstrate the utility of a viral ORFeome in identifying novel regulators of host cell immune function, which can now be expanded to other signaling pathways.