Jardetzky, T. S. | bioRxiv Science

bioRxiv2020

A bottom-up approach for the de novo design of functional proteins

De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.

bioengineering↗

bioRxiv2019

Trivalent cocktail of de novo designed immunogens enables the robust induction and focusing of functional antibodies in vivo

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a novel protein design algorithm, termed TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. Both in mice and non-human primates, cocktails of three de novo designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused pre-existing antibody responses towards defined neutralization epitopes. Overall, our de novo design approach opens the possibility of targeting specific epitopes for vaccine and therapeutic antibody development, and more generally will be applicable to design de novo proteins displaying complex functional motifs.

bioengineering↗

bioRxiv2019

Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

Kaposis sarcoma-associated herpesvirus (KSHV) is a human {gamma}-herpesvirus associated with the development of Kaposis sarcoma (KS). KSHV target cells include endothelial cells, B cells, monocytes, epithelial cells, dendritic cells, macrophages, and fibroblasts. KSHV entry into target cells is a complex multistep process and is initiated by the binding and interaction of viral envelope glycoproteins with the cellular receptors. In our current studies, we have found that EphA4 promotes KSHV gH/gL-mediated fusion and infection better than EphA2 in HEK293T cells indicating that EphA4 is a new KSHV entry receptor. To confirm that epithelial cells express EphA2 and EphA4, we analyzed the expression of EphA2 and EphA4 in epithelial cells, endothelial cells, B cells, monocytes, fibroblasts using RNA-seq data analysis of existing data sets. We found these cell types broadly express both EphA2 and EphA4 with the exception of monocytes and B cells. To confirm EphA4 is important for KSHV fusion and infection, we generated EphA2 and EphA4 single and double knockout cells. We found that both EphA2 and EphA4 play a role in KSHV fusion and infection, since EphA2/EphA4 double knockout cells had the greatest decrease in fusion activity and infection compared to single knockout cells. Fusion and infection of KSHV was rescued in the EphA2/EphA4 double knock cells upon overexpression of EphA2 and/or EphA4. EphA2 binds to both EBV and KSHV gH/gL; however, EphA4 binds only to KSHV gH/gL. Taken together, our results identify EphA4 as a new entry receptor for KSHV.\n\nImportanceThe overall entry mechanism for herpesviruses is not completely known including that for the human {gamma}-herpesviruses Kaposis sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). To fully understand the herpesvirus entry process, functional receptors need to be identified. In our current study, we found that EphA4 can also function for a KSHV entry receptor along with EphA2. Interestingly, we found that EphA4 does not function as an entry receptor for EBV whereas EphA2 does. The discovery of EphA4 as KSHV entry receptor has important implications for KSHV pathogenesis in humans and may prove useful in understanding the unique pathogenesis of KSHV infection in humans and may uncover new potential targets that can be used for the development of novel interventional strategies.

microbiology↗