New Short RNA Motifs Potentially Relevant in the SARS-CoV-2 Genome.

Background: The coronavirus disease has led to an exhaustive exploration of the SARS-CoV-2 genome. Despite the amount of information accumulated, the prediction of short RNA motifs encoding peptides mediating protein-protein or protein-drug interactions has received limited attention. Objective: The study aims to predict short RNA motifs that are interspersed in the SARS-CoV-2 genome. Methods: A method in which 14 trinucleotide families, each characterized by being composed of triplets with identical nucleotides in all possible configurations, was used to find short peptides with biological relevance. The novelty of the approach lies in using these families to search how they are distributed across genomes of different CoV genera and then to compare the distributions of these families with each other. Results: We identified distributions of trinucleotide families in different CoV genera and also how they are related, using a selection criterion that identified short RNA motifs. The motifs were reported to be conserved in SARS-CoVs; in the remaining CoV genomes analysed, motifs contained, exclusively, different configurations of the trinucleotides A, T, G and A, C, G. Eighty-eight short RNA motifs, ranging in length from 12 to 49 nucleotides, were found: 50 motifs in the 1a polyprotein-encoding orf, 27 in the 1b polyprotein-encoding orf, 5 in the spike-encoding orf, and 6 in the nucleocapsid-encoding orf. Although some motifs (~27%) were found to be intercalated or attached to functional peptides, most of them have not yet been associated with any known functions. Conclusion: Some of the trinucleotide family distributions in different CoV genera are not random; they are present in short peptides that, in many cases, are intercalated or attached to functional sites of the proteome.

Electrostatic Screening, Acidic pH and Macromolecular Crowding Increase the Self-Assembly Efficiency of the Minute Virus of Mice Capsid In Vitro.

The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers.

A Genetically Engineered, Chain Mail-Like Nanostructured Protein Material with Increased Fatigue Resistance and Enhanced Self-Healing.

Protein-based nanostructured materials are being developed for many biomedical and nanotechnological applications. Despite their many desirable features, protein materials are highly susceptible to disruption by mechanical stress and fatigue. This study is aimed to increase fatigue resistance and enhance self-healing of a natural protein-based supramolecular nanomaterial through permanent genetic modification. The authors envisage the conversion of a model nanosheet, formed by a regular array of noncovalently bound human immunodeficiency virus capsid protein molecules, into a supramolecular "chain mail." Rationally engineered mutations allow the formation of a regular network of disulfide bridges in the protein lattice. This network links each molecule in the lattice to each adjacent molecule through one covalent bond, analogous to the rivetting of interlinked iron rings in the chain mail of a medieval knight. The engineered protein nanosheet shows greatly increased thermostability and resistance to mechanical stress and fatigue in particular, as well as enhanced self-healing, without undesirable stiffening compared to the original material. The results provide proof of concept for a genetic design to permanently increase fatigue resistance and enhance self-healing of protein-based nanostructured materials. They also provide insights into the molecular basis for fatigue of protein materials.

Evolutionary conserved compositional structures hidden in genomes of the foot-and-mouth disease virus and of the human rhinovirus.

Picornaviridae family includes several viruses of great economic and medical importance. Among all members of the family we focused our attention on the human rhinovirus, the most important etiologic agent of the common cold and on the foot-and-mouth disease virus that cause of an economically important disease in cattle. Despite the low sequence similarity of the polyprotein coding open reading frames of these highly divergent picornaviruses, they have in common structural and functional similarities including a similar genomic organization, a capsid structure composed of 60 copies of four different proteins, or 3D-structures showing similar general topology, among others. We hypothesized that such similarities could be reflected in emergent common compositional structures interspersed in their genomes which were not observed heretofore. Using a methodology categorizing nucleotide triplets by their gross-composition we have found two human rhinoviruses sharing compositional structures interspersed along their genomic RNA with three foot-and-mouth disease viruses. The shared compositional structures are in one case composed by nucleotide triplets containing all nearest-neighbours of A and G and in other case containing all nearest-neighbours of A, and C. The structures are under strong evolutionary constraints for variability, allowing the access to novel viral genomic motifs with likely biological relevance. The conserved fragments would be useful to predict critical mutation points sites important from the evolutionary point of view.

Structural Analysis of a Temperature-Induced Transition in a Viral Capsid Probed by HDX-MS.

Icosahedral viral capsids are made of a large number of symmetrically organized protein subunits whose local movements can be essential for infection. In the capsid of the minute virus of mice, events required for infection that involve translocation of peptides through capsid pores are associated with a subtle conformational change. In vitro, this change can be reversibly induced by overcoming the energy barrier through mild heating of the capsid, but little is known about the capsid regions involved in the process. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry to analyze the dynamics of the minute virus of mice capsid at increasing temperatures. Our results indicate that the transition associated with peptide translocation involves the structural rearrangement of regions distant from the capsid pores. These alterations are reflected in an increased dynamics of some secondary-structure elements in the capsid shell from which spikes protrude, and a decreased dynamics in the long intertwined loops that form the large capsid spikes. Thus, the translocation events through capsid pores involve a global conformational rearrangement of the capsid and a complex alteration of its equilibrium dynamics. This study additionally demonstrates the potential of hydrogen-deuterium exchange coupled to mass spectrometry to explore in detail temperature-dependent structural dynamics in large macromolecular protein assemblies. Most importantly, it paves the way for undertaking novel studies of the relationship between structure, dynamics, and biological function in virus particles and other large protein cages.

A Method for the Annotation of Functional Similarities of Coding DNA Sequences: the Case of a Populated Cluster of Transmembrane Proteins.

The analysis of a large number of human and mouse genes codifying for a populated cluster of transmembrane proteins revealed that some of the genes significantly vary in their primary nucleotide sequence inter-species and also intra-species. In spite of that divergence and of the fact that all these genes share a common parental function we asked the question of whether at DNA level they have some kind of common compositional structure, not evident from the analysis of their primary nucleotide sequence. To reveal the existence of gene clusters not based on primary sequence relationships we have analyzed 13574 human and 14047 mouse genes by the composon-clustering methodology. The data presented show that most of the genes from each one of the samples are distributed in 18 clusters sharing the common compositional features between the particular human and mouse clusters. It was observed, in addition, that between particular human and mouse clusters having similar composon-profiles large variations in gene population were detected as an indication that a significant amount of orthologs between both species differs in compositional features. A gene cluster containing exclusively genes codifying for transmembrane proteins, an important fraction of which belongs to the Rhodopsin G-protein coupled receptor superfamily, was also detected. This indicates that even though some of them display low sequence similarity, all of them, in both species, participate with similar compositional features in terms of composons. We conclude that in this family of transmembrane proteins in general and in the Rhodopsin G-protein coupled receptor in particular, the composon-clustering reveals the existence of a type of common compositional structure underlying the primary nucleotide sequence closely correlated to function.

Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.

Do Intron and Coding Sequences of Some Human-Mouse Orthologs Evolve as a Single Unit?

It has been previously suggested that both the coding and the associated non-coding sequences of some human-mouse orthologs could evolve as a single unit. This letter deals with the observation that between mouse and humans some orthologs change significantly their compositional features as an indication that the molecular evolution is a local process. Moreover, the data shown indicate that the coding and the intron sequences of these orthologs do not evolve independently but instead both undergo a concerted evolution, evolving as a single unit, from a compositional cluster in mouse to a different compositional cluster in human.

Decrease in coccolithophore calcification and CO2 since the middle Miocene.

Marine algae are instrumental in carbon cycling and atmospheric carbon dioxide (CO2) regulation. One group, coccolithophores, uses carbon to photosynthesize and to calcify, covering their cells with chalk platelets (coccoliths). How ocean acidification influences coccolithophore calcification is strongly debated, and the effects of carbonate chemistry changes in the geological past are poorly understood. This paper relates degree of coccolith calcification to cellular calcification, and presents the first records of size-normalized coccolith thickness spanning the last 14 Myr from tropical oceans. Degree of calcification was highest in the low-pH, high-CO2 Miocene ocean, but decreased significantly between 6 and 4 Myr ago. Based on this and concurrent trends in a new alkenone ɛp record, we propose that decreasing CO2 partly drove the observed trend via reduced cellular bicarbonate allocation to calcification. This trend reversed in the late Pleistocene despite low CO2, suggesting an additional regulator of calcification such as alkalinity.

Biophysical analysis of the MHR motif in folding and domain swapping of the HIV capsid protein C-terminal domain.

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.

Association equilibrium of the HIV-1 capsid protein in a crowded medium reveals that hexamerization during capsid assembly requires a functional C-domain dimerization interface.

Polymerization of the intact capsid protein (CA) of HIV-1 into mature capsidlike particles at physiological ionic strength in vitro requires macromolecularly crowded conditions that approach those inside the virion, where the mature capsid is assembled in vivo. The capsid is organized as a hexameric lattice. CA subunits in each hexamer are connected through interfaces that involve the CA N-terminal domain (NTD); pairs of CA subunits belonging to different hexamers are connected through a different interface that involves the C-terminal domain (CTD). At physiological ionic strength in noncrowded conditions, CA subunits homodimerize through this CTD-CTD interface, but do not hexamerize through the other interfaces (those involving the NTD). Here we have investigated whether macromolecular crowding conditions are able to promote hexamerization of the isolated NTD and/or full-length CA (with an inactive CTD-CTD interface to prevent polymerization). The oligomerization state of the proteins was determined using analytical ultracentrifugation in the absence or presence of high concentrations of an inert macromolecular crowding agent. Under the same conditions that promoted efficient assembly of intact CA dimers, neither NTD nor CA with an inactive CTD-CTD interface showed any tendency to form hexamers or any other oligomer. This inability to hexamerize was observed even in macromolecularly crowded conditions. The results indicate that a functional CTD-CTD interface is strictly required for hexamerization of HIV-1 CA through the other interfaces. Together with previous results, these observations suggest that establishment of NTD-CTD interactions involved in CA hexamerization during mature HIV-1 capsid assembly requires a homodimerization-dependent conformational switching of CTD.

Analysis of calcium-induced conformational changes in calcium-binding allergens and quantitative determination of their IgE binding properties.

The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

Molecular recognition in the human immunodeficiency virus capsid and antiviral design.

Many compounds able to interfere with HIV-1 infection have been identified; some 25 of them have been approved for clinical use. Current anti-HIV-1 therapy involves the use of drug cocktails, which reduces the probability of virus escape. However, many issues remain, including drug toxicity and the emergence of drug-resistant mutant viruses, even in treated patients. Therefore, there is a constant need for the development of new anti-HIV-1 agents targeting other molecules in the viral cycle. The capsid protein CA plays a key role in many molecular recognition events during HIV-1 morphogenesis and uncoating, and is eliciting increased interest as a promising target for antiviral intervention. This article provides a structure-based, integrated review on the CA-binding small molecules and peptides identified to date, and their effects on virus capsid assembly and stability, with emphasis on recent results not previously reviewed. As a complement, we present novel experimental results on the development and proof-of-concept application of a combinatorial approach to study molecular recognition in CA and its inhibition by peptide compounds.

Molecular cloning and characterization of Cup a 4, a new allergen from Cupressus arizonica.

Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4.

Results of a pilot trial of immunotherapy with dendritic cells pulsed with autologous tumor lysates in patients with advanced cancer.

AIMS AND BACKGROUND: The purpose of the study was to test the immunological and clinical effects of infusions of dendritic cells pulsed with autologous tumor lysate in patients with advanced cancer. PATIENTS AND METHODS: Peripheral blood mononuclear cells from 15 patients with metastatic cancer (melanoma in 10, lung cancer in 2, renal cell carcinoma in 1, sarcoma in 1, breast cancer in 1) were harvested by leukapheresis after mobilization with GM-CSF (5 microg/kg/day s.c. for 4 days). Mononuclear cells were separated and cultured in GM-CSF (1000 U/ml) and interleukin-4 (1000 U/ml) for 7 days. Phenotype was assessed by 2-color flow cytometry and immunocytochemistry. On day 6, dendritic cells were pulsed with 1 g of fresh autologous tumor lysate for 24 h and infused intravenously. Interleukin-2 (6 million IU), interferon a (4 million IU) and GM-CSF (400 microg) were injected s.c. daily for 10 days beginning on the day of dendritic cell infusion. Treatment was repeated every 21 days for 3 courses. RESULTS: The morphology, immunocytochemistry and phenotype of cultured cells was consistent with dendritic cells: intense positivity for HLA-DR and CD86, with negativity for markers of other lineages, including CD3, CD4, CD8 and CD14. More than 5 x 10(7) dendritic cells were injected in all patients. Nine patients developed >5 mm delayed type cutaneous hypersensitivity reactions to tumor lysate+/-GM-CSF after the first immunization (larger than GM-CSF in all cases). Median delayed type cutaneous hypersensitivity to lysate +/- GM-CSF was 3 cm after the third immunization. One melanoma patient with skin, liver, lung and bone metastases had a partial response lasting 8 months (followed by progression in the brain). Seven patients had stable disease for >3 months, and 7 had progression. CONCLUSIONS: Infusion of tumor lysate-pulsed dendritic cells induces a strong cell-mediated antitumor immune reaction in patients with advanced cancer and has some clinical activity.

Thermodynamic stability of the C-terminal domain of the human inducible heat shock protein 70.

The stability of the substrate-binding region of human inducible Hsp70 was studied by a combination of spectroscopic and calorimetric methods. Thermal denaturation of the protein involves four accessible states: the native state, two largely populated intermediates, and the denatured state, with transition temperatures of 52.8, 56.2 and 71.2 degrees C, respectively, at pH 6.5. The intermediate spectroscopic properties resemble those of molten globules but they still retain substantial enthalpy and heat capacity of unfolding. Moreover, the similar heat capacities of the first intermediate and the native state suggests that the hydrophobic core of the intermediate would be highly native-like and that its formation would involve an increased disorder in localized portions of the structure rather than formation of a globally disordered state. The structure of the C-terminal of Hsp70 is destabilized as the pH separates from neutrality. The intermediates become populated under heat shock conditions at acidic and basic pHs. Denaturation by guanidine chloride also indicated that the protein undergoes a sequential unfolding process. The free energy change associated to the loss of secondary structure at 20 degrees C (pH 6.5) is 3.1 kcal.mol(-1) at high salt conditions. These values agree with the free energy changes estimated from differential scanning calorimetry for the transition between the second intermediate and the final denatured state.

Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions.

The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2 x 10(-4) to 6 x 10(-6) M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4-5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.