De-Novo Protein Design Strategies for Targeting Rotavirus Proteins: Evolutionary Conservation and Therapeutic Implications

During de novo emergence, new protein coding genes emerge from previously nongenic sequences. The de novo proteins they encode are dissimilar in composition and predicted biochemical properties to conserved proteins. However, functional de novo proteins indeed exist. Both identification of functional de novo proteins and their structural characterization are experimentally laborious. To identify functional and structured de novo proteins in silico, we applied recently developed machine learning based tools and found that most de novo proteins are indeed different from conserved proteins both in their structure and sequence. However, some de novo proteins are predicted to adopt known protein folds, participate in cellular reactions, and to form biomolecular condensates. Apart from broadening our understanding of de novo protein evolution, our study also provides a large set of testable hypotheses for focused experimental studies on structure and function of de novo proteins in Drosophila.

Group A rotavirus (RV) strains are a major cause of acute gastroenteritis (AGE) in infants and young children worldwide [1]. RV disease accounts for more than one-third of all diarrhea-related hospitalizations and 500,000–600,000 deaths per year [2–4]; most deaths occur in sub-Saharan Africa and Asia [3, 4]. Direct medical and indirect annual costs associated with RV disease are estimated to be €400 million in Europe [5–7] and to exceed US$ 1 billion in the United States [8]. RV strains form a genus of the Reoviridae family and possess a genome of 11 segments of double-stranded (ds) RNA, encoding 6 structural viral proteins (VPs) and 6 nonstructural proteins (NSPs). The infectious particle (ie, virion) consists of 3 layers: the inner layer (core) contains the viral genome, the viral RNA-dependent RNA polymerase (RdRp, VP1), the capping enzyme (VP3), and the scaffolding protein (VP2); the core is surrounded by a middle layer (VP6), and the outer layer consists of VP7 and VP4 [9]. RV infects mature enterocytes in the small intestine. Viral replication leads to increased intracellular Ca2+ level (effected by NSP4), increased Cl- secretion, and shut-off of host cell protein synthesis (effected by NSP3), resulting in acute osmotic and secretory diarrhea (described in [9]). Various RV genes have been implicated in the pathogenesis of AGE [10]. After RV infection, a viremic stage of, at present, unclear significance has been identified in humans and experimental animals [11–13]. The RV-encoded NSP1 blocks interferon (IFN) production by various pathways [14–17]. RV infection down-regulates the IFN- and pro-inflammatory cytokine–associated pathways in calves [18]. RV strains have a high genomic and antigenic diversity and are classified into at least 7 different groups (A–G), distinguished by different VP6. Most human RV infections are caused by group A RV strains, which are further subdivided into at least 2 subgroups (I, II), 23 G types (determined by VP7, a glycoprotein), and 31 P types (determined by VP4, a protease-sensitive protein) [9, 19–21]. RV strains with different G and P types cocirculate and change in geographical regions over time [22–25]. In temperate climate regions, most cocirculating RV strains are types G1–G4 and G9 (typically G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8], and G9P1A[8]), but other G types (G5, G8, G10, and G12), in combination with various P types, may be most prevalent in tropical areas [21, 23, 24]. Nonspecific (innate) and acquired virus-specific humoral and cellular immune responses are elicited by RV infection [26, 27] or RV vaccination [28–33]. Although currently licensed vaccines are highly efficacious in protecting children from severe RV AGE, the molecular mechanisms of protection are not fully understood. This article considers the immune responses to natural RV infection and RV vaccination in both experimental animals and humans as potential correlates of protection.

The design of de novo proteins which ensures the desirable three-dimensional (3-D) structures is the best way to test our understanding of the principles of protein structure and protein folding. The first successful engineering of a de novo protein, namely four-helix bundle, has been reported seven years ago (Regan and DeGrado, 1988) and then a number of attempts have been made in this direction during the past years (for a recent review see Sander, 1994). Most of the designed structures mimicked the 3-D structures of some natural proteins. In 1988 four of us have reported a design of a de novo protein albebetin (Figure 1) with a predetermined 3-D fold consisting of a four-stranded antiparallel β-sheet with one side screened by two α-helices (Finkelstein et al., 1988). This fold does not contradict any structural rule but has been never observed up to now in natural proteins. It consists of two identical αββ units, therefore we have called our de novo protein albebetin. We have expressed albebetin in a cell-free translational system and in E.coli and investigated it by various physical and chemical techniques (Dolgikh et al., 1991, 1992; Fedorov et al., 1992; Chemeris et al., 1994). It has been shown that the de novo protein is relatively stable and nearly as compact as natural proteins, it unfolds cooperatively at high urea concentrations and possess structural features of a definite structure consistent with the predesigned one. An eight amino acids biologically active human interferon fragment was attached to albebetin in order to obtain a de novo protein with a grafted biological function (Dolgikh et al., 1993).

Recent advances in nonrational and part-rational approaches to de novo peptide/protein design have shown increasing potential for development of novel peptides and proteins of therapeutic use. We demonstrated earlier the usefulness of one such approach recently developed by us, called "codon shuffling," in creating stand-alone de novo protein libraries from which bioactive proteins could be isolated. Here, we report the synthesis and selection of codon-shuffled de novo proteins that bind to a selected Mycobacterium tuberculosis protein target, the histone-like protein HupB, believed to be essential for mycobacterial growth. Using a versatile bacterial two-hybrid system that entailed utilization of HupB and various codon-shuffled protein libraries as bait and prey, respectively, we were able to identify proteins that bound strongly to HupB. The observed interaction was also confirmed using an in vitro assay. One of the protein binders was expressed in Mycobacterium smegmatis and was shown to appreciably affect growth in the exponential phase, a period wherein HupB is selectively expressed. Furthermore, the transcription profile of hupB gene showed a significant reduction in the transcript quantity in mycobacterial strains expressing the protein binder. Electron microscopy of the affected mycobacteria elaborated on the extent of cell damage and hinted towards a cell division malfunction. It is our belief that a closer inspection of the obtained de novo proteins may bring about the generation of small-molecule analogs, peptidomimetics, or indeed the proteins themselves as realistic leads for drug candidates. Furthermore, our strategy is adaptable for large-scale targeting of the essential protein pool of Mycobacterium tuberculosis and other pathogens.

Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Species A rotaviruses (RVs) are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years. Currently available RV vaccines were adapted from wild-type RV strains by serial passage of cultured cells or by reassortment between human and animal RV strains. These traditional methods require large-scale screening and genotyping to obtain vaccine candidates. Reverse genetics is a tractable, rapid, and reproducible approach to generating recombinant RV vaccine candidates carrying any VP4 and VP7 genes that provide selected antigenicity. Here, we developed a vaccine platform by generating recombinant RVs carrying VP4 (P[4] and P[8]), VP7 (G1, G2, G3, G8, and G9), and/or VP6 genes cloned from human RV clinical samples using the simian RV SA11 strain (G3P[2]) as a backbone. Neutralization assays using monoclonal antibodies and murine antisera revealed that recombinant VP4 and VP7 monoreassortant viruses exhibited altered antigenicity. However, replication of VP4 monoreassortant viruses was severely impaired. Generation of recombinant RVs harboring a chimeric VP4 protein for SA11 and human RV gene components revealed that the VP8* fragment was responsible for efficient infectivity of recombinant RVs. Although this system must be improved because the yield of vaccine viruses directly affects vaccine manufacturing costs, reverse genetics requires less time than traditional methods and enables rapid production of safe and effective vaccine candidates.IMPORTANCE Although vaccines have reduced global RV-associated hospitalization and mortality over the past decade, the multisegmented genome of RVs allows reassortment of VP4 and VP7 genes from different RV species and strains. The evolutionary dynamics of novel RV genotypes and their constellations have led to great genomic and antigenic diversity. The reverse genetics system is a powerful tool for manipulating RV genes, thereby controlling viral antigenicity, growth capacity, and pathogenicity. Here, we generated recombinant simian RVs (strain SA11) carrying heterologous VP4 and VP7 genes cloned from clinical isolates and showed that VP4- or VP7-substituted chimeric viruses can be used for antigenic characterization of RV outer capsid proteins and as improved seed viruses for vaccine production.

Rotavirus (RV) and norovirus (NoV) are considered the most common causes of viral gastroenteritis in children. In this study, the prevalence of RV and NoV infection in 55 children with diarrhea from the rural community Akinlalu in Southwestern Nigeria was investigated using real-time PCR assays. The RV and NoV strains were genotyped by PCR and/or sequencing. RV and NoV infections occurred with a prevalence of 34.5% and 25.5% respectively, with predominance in children <1 year. Most infections occurred during the dry season with increasing prevalence of RV as the dry season progressed (October-January). Infections with RV VP6 subgroup (SG) II were more prevalent (27.3%) than SGI (7.3%). Similarly, NoV genogroup II infections were more common (23.6%) than genogroup I (1.8%). Five children out of 55 (9.1%) were co-infected with both RV and NoV. Notably, G12P[8] was the predominant RV strain (36.8%, n = 7), observed for the first time in Nigeria. The VP7 gene of the G12 strains clustered within lineage III, sharing high nucleotide identity with each other (>99%) indicating introduction in Nigeria from a single donor. Furthermore, a putative novel genotype within genogroup I NoV was detected, which till date has only been reported once in humans. To conclude, a high prevalence of the emerging G12P[8] RV strain was observed for the first time in Nigeria, as well as a putative novel NoV genotype in humans. These results provide new information which can be important for future vaccine evaluations and possible introduction in Nigeria.

The changing epidemiology of group A rotavirus (RV) strains in humans and swine, including emerging G9 strains, poses new challenges to current vaccines. In this study, we comparatively assessed the pathogenesis of porcine RV (PRV) G9P[13] and evaluated the short-term cross-protection between this strain and human RV (HRV) Wa G1P[8] in gnotobiotic pigs. Complete genome sequencing demonstrated that PRV G9P[13] possessed a human-like G9 VP7 genotype but shared higher overall nucleotide identity with historic PRV strains. PRV G9P[13] induced longer rectal virus shedding and RV RNAemia in pigs than HRV Wa G1P[8] and generated complete short-term cross-protection in pigs challenged with HRV or PRV, whereas HRV Wa G1P[8] induced only partial protection against PRV challenge. Moreover, PRV G9P[13] replicated more extensively in porcine monocyte-derived dendritic cells (MoDCs) than did HRV Wa G1P[8]. Cross-protection was likely not dependent on serum virus-neutralizing (VN) antibodies, as the heterologous VN antibody titers in the sera of G9P[13]-inoculated pigs were low. Thus, our results suggest that heterologous protection by the current monovalent G1P[8] HRV vaccine against emerging G9 strains should be evaluated in clinical and experimental studies to prevent further dissemination of G9 strains. Differences in the pathogenesis of these two strains may be partially attributable to their variable abilities to replicate and persist in porcine immune cells, including dendritic cells (DCs). Additional studies are needed to evaluate the emerging G9 strains as potential vaccine candidates and to test the susceptibility of various immune cells to infection by G9 and other common HRV/PRV genotypes. The changing epidemiology of porcine and human group A rotaviruses (RVs), including emerging G9 strains, may compromise the efficacy of current vaccines. An understanding of the pathogenesis and genetic, immunological, and biological features of the new emerging RV strains will contribute to the development of new surveillance and prevention tools. Additionally, studies of cross-protection between the newly identified emerging G9 porcine RV strains and a human G1 RV vaccine strain in a susceptible host (swine) will allow evaluation of G9 strains as potential novel vaccine candidates to be included in porcine or human vaccines.

BackgroundThe eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery.ResultsUsing extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the β' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi β-barrel (DPBB) domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP β' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases. In addition to the β' subunit, β subunit of DDRP also contains a DPBB domain, which is, however, distorted by large inserts and does not harbor a counterpart of the DbDGD motif. The DPBB domains of the two DDRP subunits together form the catalytic cleft, with the domain from the β' subunit supplying the metal-coordinating DbDGD motif and the one from the β subunit providing two lysine residues involved in catalysis. Given that the two DPBB domains of DDRP contribute completely different sets of active residues to the catalytic center, it is hypothesized that the ultimate ancestor of RNA polymerases functioned as a homodimer of a generic, RNA-binding DPBB domain. This ancestral protein probably did not have catalytic activity and served as a cofactor for a ribozyme RNA polymerase. Subsequent evolution of DDRP and RDRP involved accretion of distinct sets of additional domains. In the DDRPs, these included a RNA-binding Zn-ribbon, an AT-hook-like module and a sandwich-barrel hybrid motif (SBHM) domain. Further, lineage-specific accretion of SBHM domains and other, DDRP-specific domains is observed in bacterial DDRPs. In contrast, the orthologs of the β' subunit in archaea and eukaryotes contains a four-stranded α + β domain that is shared with the α-subunit of bacterial DDRP, eukaryotic DDRP subunit RBP11, translation factor eIF1 and type II topoisomerases. The additional domains of the RDRPs remain to be characterized.ConclusionsEukaryotic RNA-dependent RNA polymerases share the catalytic double-psi β-barrel domain, containing a signature metal-coordinating motif, with the universally conserved β' subunit of DNA-dependent RNA polymerases. Beyond this core catalytic domain, the two classes of RNA polymerases do not have common domains, suggesting early divergence from a common ancestor, with subsequent independent domain accretion. The β-subunit of DDRP contains another, highly diverged DPBB domain. The presence of two distinct DPBB domains in two subunits of DDRP is compatible with the hypothesis that the ultimate ancestor of RNA polymerases was a RNA-binding DPBB domain that had no catalytic activity but rather functioned as a homodimeric cofactor for a ribozyme polymerase.

Emergence of Rotavirus P2A[6] Genotype Circulating in the Americas

In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data.

Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate these questions, we have created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. Previously designed libraries contained numerous functional proteins. However, they often formed dynamic, rather than well-ordered structures, which complicated structural and mechanistic characterization. To address this challenge, we describe the development of new libraries based on the de novo protein S-824, a 4-helix bundle with a very stable 3-dimensional structure. Distinct from previous libraries, we targeted variability to a specific region of the protein, seeking to create potential functional sites. By characterizing variant proteins from this library, we demonstrate that the S-824 scaffold tolerates diverse amino acid substitutions in a putative cavity, including buried polar residues suitable for catalysis. We designed and created a DNA library encoding 1.7 × 106 unique protein sequences. This new library of stable de novo α-helical proteins is well suited for screens and selections for a range of functional activities in vitro and in vivo.

Simple SummaryAlmost half of the protein ingested by broilers is not retained and is excreted, impairing the nitrogen utilization, health and productivity of the animals, and intensifying the environmental impact of poultry meat production. This work proposes two potential tools, combining traditional nutrition with biotechnological, metabolomics, computational and protein engineering knowledge, which can contribute to improving precise amino acid nutrition in broilers in the future: (i) the use of serum uric nitrogen content as a rapid biomarker of amino acid imbalances, and (ii) the design and modeling of de novo proteins that are fully digestible and fit exactly to the animal’s requirements. Both tools can open up new opportunities to form an integrated framework for precise amino acid nutrition in broilers, helping us to achieve more efficient, resilient, and sustainable production. This information can help to determine the exact ratio of amino acids that will improve the efficiency of the use of nitrogen by poultry.Precision nutrition in broilers requires tools capable of identifying amino acid imbalances individually or in groups, as well as knowledge on how more digestible proteins can be designed for innovative feeding programs adjusted to animals’ dynamic requirements. This work proposes two potential tools, combining traditional nutrition with biotechnological, metabolomic, computational and protein engineering knowledge, which can contribute to improving the precise amino acid nutrition of broilers in the future: (i) the use of serum uric nitrogen content as a rapid biomarker of amino acid imbalances, and (ii) the design and modeling of de novo proteins that are fully digestible and fit exactly to the animal’s requirements. Each application is illustrated with a case study. Case study 1 demonstrates that serum uric nitrogen can be a useful rapid indicator of individual or group amino acid deficiencies or imbalances when reducing dietary protein and adjusting the valine and arginine to lysine ratios in broilers. Case study 2 describes a stepwise approach to design an ideal protein, resulting in a potential amino acid sequence and structure prototype that is ideally adjusted to the requirements of the targeted animal, and is theoretically completely digestible. Both tools can open up new opportunities to form an integrated framework for precise amino acid nutrition in broilers, helping us to achieve more efficient, resilient, and sustainable production. This information can help to determine the exact ratio of amino acids that will improve the efficiency of the use of nitrogen by poultry.

Phylogenetic analysis of porcine rotavirus in Argentina: Increasing diversity of G4 strains and evidence of interspecies transmission