Physics-based approach to extend a de novo TIM barrel with rationally designed helix-loop-helix motifs.

Computational protein design promises the ability to build tailor-made proteins de novo. While a range of de novo proteins have been constructed so far, the majority of these designs have idealized topologies that lack larger cavities which are necessary for the incorporation of small molecule binding sites or enzymatic functions. One attractive target for enzyme design is the TIM-barrel fold, due to its ubiquity in nature and capability to host versatile functions. With the successful de novo design of a 4-fold symmetric TIM barrel, sTIM11, an idealized, minimalistic scaffold was created. In this work, we attempted to extend this de novo TIM barrel by incorporating a helix-loop-helix motif into its ßα-loops by applying a physics-based modular design approach using Rosetta. Further diversification was performed by exploiting the symmetry of the scaffold to integrate two helix-loop-helix motifs into the scaffold. Analysis with AlphaFold2 and biochemical characterization demonstrate the formation of additional α-helical secondary structure elements supporting the successful extension as intended.

Analysis of PERV-C superinfection resistance using HA-tagged viruses.

BACKGROUND: Using pigs as organ donors has advanced xenotransplantation to the point that it is almost ready for clinical use. However, there is still a zoonotic risk associated with xenotransplantation, and the potential transmission of porcine endogenous retroviruses needs to be surveyed. Despite significant attempts to eliminate this risk, by the selection of PERV-C free pigs with low expression of PERV-A, -B, and by the genome-wide inactivation of PERV using CRISPR/Cas9, the impact of superinfection resistance (SIR) was not investigated. SIR is a viral trait that prevents reinfection (superinfection). For PERV, the underlying mechanism is unclear, whether and how cells, that harbor functional PERV, are protected. Using PERV-C(5683) as a reference virus, we investigated SIR in a newly developed in vitro model to pursue the mechanism and confirm its protective effect. RESULTS: We developed three PERV-C constructs on the basis of PERV-C(5683), each of which carries a hemagglutinin tag (HA-tag) at a different position of the envelope gene (SP-HA, HA-VRA, and RPep-HA), to distinguish between primary infection and superinfection. The newly generated PERV-C(5683)-HA viruses were characterized while quantifying the viral RNA, reverse transcriptase activity, protein expression analysis, and infection studies. It was demonstrated that SP-HA and RPep-HA were comparable to PERV-C(5683), whereas HA-VRA was not replication competent. SP-HA and RPep-HA were chosen to challenge PERV-C(5683)-positive ST-IOWA cells demonstrating that PERV-C-HA viruses are not able to superinfect those cells. They do not integrate into the genome and are not expressed. CONCLUSIONS: The mechanism of SIR applies to PERV-C. The production of PERV-C particles serves as a defense mechanism from superinfection with exogenous PERV-C. It was demonstrated by newly generated PERV-C(5683)-HA clones that might be used as a cutting-edge tool. The HA-tagging of PERV-C is novel, providing a blueprint for the tagging of other human tropic PERV viruses. The tagged viruses are suitable for additional in vitro and in vivo infection studies and will contribute, to basic research on viral invasion and pathogenesis. It will maintain the virus safety of XTx.