Strategies for bacterial expression of protein-peptide complexes: application to solubilization of papillomavirus E6

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.     

Mosquito survey in Mauritania: Detection of Rift Valley fever virus and dengue virus and the determination of feeding patterns

In Mauritania, several mosquito-borne viruses have been reported that can cause devastating diseases in animals and humans. However, monitoring data on their occurrence and local distribution are limited. Rift Valley fever virus (RVFV) is an arthropod-borne virus that causes major outbreaks throughout the African continent and the Arabian Peninsula. The first Rift Valley fever (RVF) epidemic in Mauritania occurred in 1987 and since then the country has been affected by recurrent outbreaks of the disease. To gain information on the occurrence of RVFV as well as other mosquito-borne viruses and their vectors in Mauritania, we collected and examined 4,950 mosquitoes, belonging to four genera and 14 species. The mosquitoes were captured during 2018 in the capital Nouakchott and in southern parts of Mauritania. Evidence of RVFV was found in a mosquito pool of female Anopheles pharoensis mosquitoes collected in December on a farm near the Senegal River. At that time, 37.5% of 16 tested Montbéliarde cattle on the farm showed RVFV-specific IgM antibodies. Additionally, we detected IgM antibodies in 10.7% of 28 indigenous cattle that had been sampled on the same farm one month earlier. To obtain information on potential RVFV reservoir hosts, blood meals of captured engorged mosquitoes were analyzed. The mosquitoes mainly fed on humans (urban areas) and cattle (rural areas), but also on small ruminants, donkeys, cats, dogs and straw-colored fruit bats. Results of this study demonstrate the circulation of RVFV in Mauritania and thus the need for further research to investigate the distribution of the virus and its vectors. Furthermore, factors that may contribute to its maintenance should be analyzed more closely. In addition, two mosquito pools containing Aedes aegypti and Culex quinquefasciatus mosquitoes showed evidence of dengue virus (DENV) 2 circulation in the city of Rosso. Further studies are therefore needed to also examine DENV circulation in Mauritania.     

Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53

The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.     

Computational Design and Preparation of Cation‐Disordered Oxides for High‐Energy‐Density Li‐Ion Batteries

Cation‐disordered lithium‐excess metal oxides have recently emerged as a promising new class of high‐energy‐density cathode materials for Li‐ion batteries, but the exploration of disordered materials has been hampered by their vast and unexplored composition space. This study proposes a practical methodology for the identification of stable cation‐disordered rocksalts. Here, it is established that the efficient method, which makes use of special quasirandom structures, correctly predicts cation‐ordering strengths in agreement with accurate Monte‐Carlo simulations and experimental observations. By applying the approach to the composition space of ternary oxides with formula unit LiA_0.5B_0.5O₂ (A, B: transition metals), this study discovers a previously unknown cation‐disordered structure, LiCo_0.5Zr_0.5O₂, that may function as the basis for a new class of cation‐disordered cathode materials. This computational prediction is confirmed experimentally by solid‐state synthesis and subsequent characterization by powder X‐ray diffraction demonstrating the potential of the computational screening of large composition spaces for accelerating materials discovery.     

Omura's whale off West Africa: autochthonous population or inter-oceanic vagrant in the Atlantic Ocean?

The surveillance of marine mammal strandings within the framework of a biomonitoring scheme for the Mauritanian coast led to the discovery in 2013 of a 3.98?m long, juvenile rorqual Balaenoptera sp. in an advanced state of decomposition near Chott Boul (16°32.488′N, 016°27.0317′W). Photographs and skin samples were taken, but the specimen could not be collected. Based on limited morphological evidence, only Balaenoptera edeni and B. omurai were plausible. Sequences of three mtDNA regions, i.e. parts of the cox1 and the cytb genes as well as the D-loop, for a total of 2636?bp (> 16% of the mitogenome) identified the specimen as an Omura's whale, B. omurai, the first record in the Atlantic Ocean and at least 11,400?km away from its closest known range in the SW Indian Ocean (Madagascar). The question of whether the specimen is a vagrant or belongs to an unrecognized Atlantic population is discussed. Advection by currents or transport on a ship's bow bulb following collision are discarded. Circumstances (juvenile status, great distance from Indo-Pacific, necessary inter-oceanic passage through cold temperate waters) may slightly favour the hypothesis that B. omurai, if rare, could be autochthonous in the Atlantic. Beach surveys remain a useful tool to assess trends in cetacean species composition, to detect unusual mortality events and to help assess the impacts of anthropogenic activities. This is particularly applicable to remote areas where the marine mammal fauna is poorly known and where fisheries effort is high, such as the Mauritanian coast.     

Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization,Biophysical Characterization,and Phylogenetic Analysis

Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.Papillomaviruses (PVs) are nonenveloped, epitheliotropic, double-stranded DNA viruses that cause a variety of diseases in a multitude of hosts. Based on available whole-genome sequences and subgenomic amplicons, more than 200 human and over 55 nonhuman mammalian PV types have been described (7, 34, 35, 37, 38). To date, two avian PV types have been characterized (37, 38).The genomic organizations of the PVs are remarkably similar. The genome is ca. 8 kb in length and comprises an upstream regulatory region (URR), the early genes (E1, E2, E4, E6, and E7), and the late genes that encode the capsid proteins (L1 and L2). Although most PVs code for these seven open reading frames (ORFs), only the URR, the replicative proteins E1 and E2 (and possibly the E4 gene), and the capsid proteins L1 and L2 are strictly conserved in all PVs (11).Upon infection of the stratified squamous epithelia, PV gene expression is linked to the differentiation state of the infected epithelium cells. The expression of early PV proteins, in particular E6 and E7, primes the proliferation of the infected epithelium. This proliferation, which is absolutely required for viral replication, may become malignant depending on the PV strain considered. Several “high-risk” mucosal human PV (HPV) strains (predominantly HPV type 16 [HPV-16], HPV-18, and HPV-45) have been shown to be responsible for cervical cancer (19).The ability of PVs to induce proliferation of the infected cells has been attributed mainly to two small “oncoproteins,” E6 and E7. In genital high-risk HPVs, these proteins play a prominent role in cell immortalization and transformation (31). In most mammalian PVs, E6 is a small protein of about 150 amino acids, with two conserved N- and C-terminal zinc-binding domains, E6N and E6C, respectively (12). The solution structure of the HPV-16 E6C domain was recently determined (23). The sequence alignments pointed to a structural similarity between the E6C and E6N domains, suggesting that a single-domain protein possessing the same fold might have once existed. Earlier phylogenetic studies had suggested that gene duplication may have given rise to the current double-domain E6 proteins (5). Interestingly, although the E6 ORF has been found in most mammalian PVs (with the exception of bovine papillomavirus type 3 [BPV-3], BPV-4, BPV-6, HPV-101, and HPV-103 [3, 7]), it was not detected in the two avian PVs previously sequenced (37, 38).In this study, we present the full sequence of the genome of a novel PV from a francolin bird (Francolinus leucoscepus PV type 1 [FlPV-1]) and compare it to the two other avian PV genomes known to date (Psittacus erithacus PV [PePV] and Fringilla coelebs PV [FPV]). In light of recent structural data, we compare the unusual avian E6 and E7 ORFs to their mammalian orthologs. We describe the expression and purification of recombinant avian PV E6 and E7 proteins, their biophysical characterization, and cellular localization. Finally, we use phylogenetic techniques to investigate the evolutionary history of the E6 protein family.