Structural analysis of ORF4b protein of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in complex with host proteins and its role in innate immunity

Middle Eastern respiratory syndrome-related coronavirus (MERS-CoV) is a highly pathogenic emerging zoonotic, non-segmented, positive sense RNA virus related to severe acute respiratory syndrome-related coronavirus (SARS-CoV) 1 and SARS-CoV 2. It belongs to the subgenus Merbecovirus of the genus Betacoronavirus. The MERS-CoV ORF4b protein is a viral accessory protein that functions as an innate immune inhibitor and is localized into the nucleus. These viral proteins are translocated by host proteins such as IMPAs and IMPBs. ORF4b protein has been shown to bind to both IMPAs and IMPBs. This study reveals an unusual binding mechanism of MERS-CoV ORF4b with IMPA with high resolution structures of the nuclear localisation signal (NLS) of ORF4b bound to two distinct IMPA family members. In both cases, the canonical P2 site, conserved almost exclusively for a Lys residue, is occupied by an unbound Val in MERS ORF4b. The P2 Lys is responsible for ~50% of binding strength of canonical binding mechanism. This binding loss is compensated by a Arg at the P0 site in the MERS-CoV ORF4b NLS. Mutations within the NLS regions of ORF4b dramatically alter the binding mechanism which returns to utilisation of the canonical P2 Lys. This study also shows that there is a clear overlap between the interfaces of the NF-kB component p50 bound to IMPA2 and IMPA3, and ORF4b bound to same IMPAs structures. This confirms that ORF4b outcompetes with p50 to bind with IMPAs, suggesting a basis for innate immune inhibition. Our current study suggests that even a small mutation could change the binding mechanism and thereby its ability to affect the innate immune signalling pathways in hosts. Compared with WT, MERS-CoV ORF4b mutation of Arg24 to Ala binds only at the major site of IMPA2, mediated through a canonical Lys at the P2 site. MERS-CoV ORF4b His26 to Ala mutation shows an identical binding pattern with MERS-CoV WT ORF4b NLS. Mutation at Arg33 to Ala binds in a similar way to the Arg24 mutant, with Lys30 positioned at P2 site. MERS-CoV ORF4b Arg37 to Ala also shows similar pattern with Arg24 and Arg33 where the P2 site is occupied with a Lys. Consistent with the structural and biochemical results, cellular data shows that mutation Arg24 alone has a small effect on the nuclear import of MERS-CoV ORF4b while Arg33 alone or in combination with Arg24 has significantly reduced the nuclear localization of MERS-CoV ORF4b protein. This confirms that the novel mechanism is important for IMPA interactions, nuclear import, and inhibition of nuclear pathways. This provides insight to explore how small changes within the NLS regions through evolution in closely related coronaviruses such as HKU5, can drastically alter the binding mechanism.
Usually, NLS containing cargo proteins are transported from cytoplasm to nucleus via either binding to IMPAs or IMPA/B heterodimer (termed the non-classical and classical import pathways respectively). This study shows that MERS-CoV ORF4b can bind to both IMPA and IMPBs. This study also shows that whilst the ORF4b binding to IMPB1 is stronger than binding to IMPA (even lacking the auto-inhibitory IBB domain), ORF4b binding is not sufficiently strong to disrupt the high affinity interaction between IMPB1 and IMPA. This study also shows that ORF4b binding to IMPB1 is also not sufficient to disrupt the high affinity interaction of IMPB1 for Ran. This suggests that MERS-CoV ORF4b could bind (and inhibit) both IMPA/B and IMPB1 mediated pathway. Interestingly, this study shows MERS-CoV ORF4b can also bind with other IMPB family members such as IPO5 and TNPO1, suggesting that its capacity to inhibit a multitude of import cargo. Progress that has been made towards the interaction between MERS-CoV ORF4b and IPO5 through a low-resolution structure is also described.
This research uncovers a distinctive binding mechanism demonstrated by MERS-CoV ORF4b. This mechanism involves binding to IMPA, resulting in the suppression of the immune response. Through a systematic profiling of highly specific interactions with different IMPA isoforms and a thorough exploration of the structural foundation of IMPA's specificity, this study also sheds light on the process by which viruses selectively target IMPA isoforms. This targeting effectively hampers the host's immune signalling, all the while leaving other vital nuclear transport pathways crucial for cell survival and virus replication unaffected. Moreover, this insight hints at the potential for MERS-CoV ORF4b to engage with other host proteins, including IMPB family members. The current study implies a broader scope of host protein binding, which in turn has more extensive implications on nuclear import processes. In essence, the expanded understanding of host protein binding and its impact on nuclear import processes has far-reaching implications for virology, therapeutics, and our comprehension of cellular biology. It paves the way for innovative strategies to combat viral infections and offers valuable insights into the delicate balance between viruses and their host cells.