Peptide L13S Derived from the BRN2 POU Domain Reduces Metastasis In Vivo and Inhibits Cell Migration and Invasion In Vitro.

BACKGROUND/AIM: The Brain-Specific Homeobox/POU Domain Protein 2 (BRN2) transcription factor supports melanoma progression by regulating the expression of several genes involved in cell migration and invasion. We hypothesized that a peptide designed based on the POU domain of BRN2 could block the BRN2 transcription activity and, consequently, reduce metastasis. MATERIALS AND METHODS: Cell viability was accessed by Trypan Blue exclusion dye assay and xCelligence platform. Wound-healing scratch assay and transwell invasion with matrigel membrane assay were performed to analyze cell migration and invasion. The internalization mechanism of the L13S peptide was investigated using confocal microscopy and wound-healing scratch assay. The impact of L13S on cell protein expression was analyzed through western blotting. In vivo assays were conducted to evaluate the protective effect and toxicity of L13S in a metastatic model using murine melanoma cells. RESULTS: Here, we show that the peptide named L13S can inhibit the migration and invasion of murine melanoma cells (B16F10-Nex2) as well as the migration of human melanoma cells (SK-MEL-25 and A375) by regulating the expression of proteins involved in motility. Mechanistically, we found that L13S is internalized by murine melanoma cells via macropinocytosis and binds actin filaments and nuclei. More importantly, in vivo studies indicated that the peptide was able to significantly inhibit lung metastasis in syngeneic models without off-target effects and with virtually no cytotoxicity toward normal organs. CONCLUSION: L13S peptide is a strong candidate for further development as an anticancer agent for the treatment of melanoma metastasis.

Specialized outpatient care for maternal and child health in PlanificaSUS areas / Atenção ambulatorial especializada à saúde materno-infantil em regiões do PlanificaSUS

ABSTRACT OBJECTIVE To describe the organization of specialized outpatient clinics, according to the Secondary Outpatient Care Unit (SOCU) model according to the health care planning (HCP) methodology. METHODS This is a descriptive and cross-sectional study, which used secondary data from the PlanificaSUS project. It was carried out in 16 outpatient clinics specialized in maternal and child care, distributed in the five Brazilian geographic regions. A structured questionnaire was used for self-assessment on the implementation of 12 parameters in two moments, in 2019 and in 2020. These parameters are related to the care, educational, and supervisory functions set out in the SOCU model. RESULTS In 2019, only 37.5% (six) of the outpatient clinics completed at least one parameter related to the care function, most frequently the multiprofessional team with interdisciplinary action (completed in 18.8% of the outpatient clinics). No parameters from the educational and supervisory functions were completed at this initial stage. In 2020, on the other hand, parameters related to the care function also showed higher frequency, higlighting the use of the same criterion by primary care teams and outpatient clinics for risk stratification (completed in 68.8% of the outpatient clinics). In the educational and supervisory functions, parameters related to the encounter between primary care teams and outpatient clinics for case management development, integrated training promotion, and close communication bond among these professionals also increased. Completion of these three parameters was identified in 25%, 25%, and 37.5% of the outpatient clinics, respectively. CONCLUSIONS The planning methodology fostered reflection and discussion about the (re)organization of the work process and contributed to changes in maternal and child health care practices within specialized outpatient care, integrated with primary health care (PHC), from the perspective of care networks. We believe that such advances enhance access and equitable care for high-risk pregnant women and children in different geographical regions of Brazil.

RESUMO OBJETIVO Descrever a organização de ambulatórios especializados, conforme o modelo Ponto de Atenção Secundária Ambulatorial (Pasa), por meio da metodologia da planificação de atenção à saúde (PAS). MÉTODOS Trata-se de um estudo descritivo e transversal, que utilizou dados secundários do projeto PlanificaSUS. Foi realizado em 16 ambulatórios especializados na linha de cuidado materno infantil, distribuídos nas cinco regiões geográficas brasileiras. Um questionário estruturado foi utilizado para autoavaliação sobre a implantação de 12 parâmetros em dois momentos, em 2019 e em 2020. Esses parâmetros são relacionados às funções assistencial, educacional e de supervisão previstas no modelo Pasa. RESULTADOS Em 2019, apenas 37,5% (seis) dos ambulatórios apresentaram, pelo menos, um parâmetro concluído, sendo com maior frequência o de equipe multiprofissional com atuação interdisciplinar (concluído em 18,8% dos ambulatórios), relacionado à função assistencial. Nenhum parâmetro das funções educacional e de supervisão estava concluído nesse primeiro momento. Já em 2020, os parâmetros relacionados à função assistencial também apresentaram maior frequência, destacando-se a utilização do mesmo critério pelas equipes da atenção primária e dos ambulatórios para estratificação de risco (concluídos em 68,8% dos ambulatórios). Nas funções educacional e supervisional, os parâmetros de encontro entre as equipes da atenção primária e dos ambulatórios para o desenvolvimento da gestão de caso, promoção de capacitação integrada e vínculo estreito de comunicação entre esses profissionais também aumentaram, identificando-se conclusão destes três parâmetros em 25%, 25% e 37,5% dos ambulatórios, respectivamente. CONCLUSÕES A metodologia da planificação proporcionou reflexão e discussão acerca da (re)reorganização do processo de trabalho e contribuiu para a mudanças de práticas de cuidado à saúde materno-infantil na atenção ambulatorial especializada, de forma integrada com a atenção primária à saúde (APS), na perspectiva das redes de atenção. Acredita-se que tais avanços potencializam o acesso e cuidado equitativo de gestantes e crianças de alto risco nas diferentes regiões geográficas brasileiras.

Gravidade do pé diabético: fatores socioeconômicos em estudo envolvendo 5300 operados / Diabect foot severety: socieconomic factors in a study with 5300 patients / Gravedad del pie diabético: factores socioeconómicos en un estudio con 5300 operados

As amputações decorrentes de pé diabético são graves e geram impacto social e econômico, porém são passíveis de prevenção. O objetivo deste estudo foi testar a hipótese de que há associação entre fatores socioeconômicos e a gravidade do pé diabético. Em estudo observacional restrospectivo, foram avaliados 5.300 prontuários de pacientes portadores de pé diabético operados pelo Serviço de Cirurgia Vascular do Hospital Geral Roberto Santos (HGRS), no período de fevereiro de 2005 a dezembro de 2015. Foram coletadas as condições socioeconômicas para verificar correlação com a gravidade do pé diabético. Os dados foram tabulados no Excel e a distribuição foi analisada no GraphPad Prism 8.0, por meio do teste qui-quadrado e da técnica de regressão logística. Foram considerados valores de p < 0,05 como estatisticamente significantes. A maioria dos pacientes era de meia-idade (entre 53 e 58 anos), do sexo masculino (57,58%), com o ensino fundamental completo (50,38%) e ganhava menos de um salário mínimo (56,81%). A principal doença associada foi a hipertensão arterial sistêmica (HAS) (55,64%). Com relação à distância, foi observado que 48,15% residiam a menos de 100 km da unidade hospitalar. Observou-se prevalência de amputação menor (37,36%) e taxa de letalidade de 6,32%. Também foi possível correlacionar, quanto à amputação maior, maiores frequências nos pacientes acima de 70 anos (p = 0,045), naqueles com renda familiar abaixo de um salário mínimo (p = 0,05), nos portadores de doença arterial coronariana (Daop) (p = 0,035) e nos indivíduos que residiam a uma distância acima de 400 km do HGRS. Registrou-se maior taxa de óbito nos portadores de coronariopatia, com idade superior a 70 anos e nos submetidos à amputação maior. Diante disso, faz-se necessário treinamento de profissionais e adoção de medidas de saúde pública, visando identificar precocemente a população de maior gravidade, a fim de evitar desfechos desfavoráveis.

Amputations due to diabetic foot are serious and generate social and economic impact, but are preventable. This research sought to verify whether socioeconomic factors are associated with diabetic foot severity. A retrospective observational study evaluated 5,300 medical records from patients with diabetic foot, operated by the Vascular Surgery Service at Roberto Santos General Hospital (HGRS), between February 2005 and December 2015. Socioeconomic data were collected to verify correlation with diabetic foot severity. After tabulation in Excel, data distribution was analyzed by GraphPad Prism 8.0 using the Chi-square test and logistic regression. P-values of < 0.05 were considered statistically significant. Most patients were middle-aged (between 53 and 58 years old) men (57.58%), with basic education (50.38%) and earning less than one minimum wage (56.81%). Hypertension was the main associated disease (55.64%). Regarding distance, 48.15% of the patients lived less than 100 km from the hospital unit. Results showed prevalence of minor amputation (37.36%) and a fatality rate of 6.32%. Major amputation was associated with patients over 70 years of age (p = 0.045), family income below 1 MW (p = 0.05), patients with coronary artery disease (CAD) (p = 0.035), and individuals who lived 400 km away from the HGRS. Patients with CAD aged over 70 years and those who underwent major amputation showed a higher death rate. As such, training professionals and adopting public health measures aimed at early identification of at risk population are necessary to avoid unfavorable outcomes.

Las amputaciones por pie diabético son graves y generan impacto social y económico, pero son prevenibles. El objetivo de este estudio fue probar el hipótesis de que existe una asociación entre los factores socioeconómicos y la gravedad de pie diabético. En un estudio observacional retrospectivo, se evaluaron 5.300 historias clínicas de pacientes con pie diabético operados por el Servicio de Cirugía Vascular del Hospital General Roberto Santos (HGRS), en el período comprendido entre febrero 2005 a diciembre 2015. Se recogieron condiciones socioeconómicas para verificar una correlación con la gravedad del pie diabético. Los datos se tabularon en Excel y la distribución se analizó en GraphPad Prism 8.0 mediante la prueba de chi-cuadrado y la técnica de regresión logística. Los valores de p < 0,05 se consideraron estadísticamente significativos. La mayoría de los pacientes eran de mediana edad (entre 53 y 58 años), del sexo masculino (57,58%), con el nivel de estudios de la primaria (50,38%) y que ganaban menos de 1 salario mínimo (56,81%). La principal enfermedad asociada fue la hipertensión arterial sistémica ­HAS­ (55,64%). En cuanto a la distancia, se observó que el 48,15% vive a menos de 100 km de la unidad hospitalaria. Hubo mayor frecuencia de amputaciones menores (37,36%) y una tasa de mortalidad del 6,32%. También fue posible correlacionar la amputación mayor, mayor frecuencia en pacientes mayores de 70 años (p = 0,045), en aquellos con renta familiar inferior a 1 salario mínimo (p = 0,05), en aquellos con enfermedad arterial coronaria ­Daop­ (p = 0,035) y en personas que residen a una distancia superior a 400 km del HGRS. Hubo una mayor tasa de muerte en pacientes con enfermedad de las arterias coronarias, mayores de 70 años y sometidos a una amputación importante. Ante esto, es necesario una formación de profesionales y adopción de medidas de salud pública, con el objetivo de identificar tempranamente la población más grave y así evitar resultados desfavorables.

Structure-guided stabilization of pathogen-derived peptide-HLA-E complexes using non-natural amino acids conserves native TCR recognition.

The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions. Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs.

IAF, QGF, and QDF Peptides Exhibit Cholesterol-Lowering Activity through a Statin-like HMG-CoA Reductase Regulation Mechanism: In Silico and In Vitro Approach.

In this study, in silico approaches are employed to investigate the binding mechanism of peptides derived from cowpea ß-vignin and HMG-CoA reductase. With the obtained information, we designed synthetic peptides to evaluate their in vitro enzyme inhibitory activity. In vitro, the total protein extract and <3 kDa fraction, at 5000 µg, support this hypothesis (95% and 90% inhibition of HMG-CoA reductase, respectively). Ile-Ala-Phe, Gln-Gly-Phe, and Gln-Asp-Phe peptides were predicted to bind to the substrate binding site of HMGCR via HMG-CoAR. In silico, it was established that the mechanism of HMG-CoA reductase inhibition largely entailed mimicking the interactions of the decalin ring of simvastatin and via H-bonding; in vitro studies corroborated the predictions, whereby the HMG-CoA reductase activity was decreased by 69%, 77%, and 78%, respectively. Our results suggest that Ile-Ala-Phe, Gln-Gly-Phe, and Gln-Asp-Phe peptides derived from cowpea ß-vignin have the potential to lower cholesterol synthesis through a statin-like regulation mechanism.

Canulação biliar difícil: devemos tentar uma segunda CPRE após uma fistulotomia papilar mal-sucedida? / Difficult biliary cannulation: should we always try a second ercp after a failed needle-knife fistulotomy?

ABSTRACT BACKGROUND: A successful bile duct cannulation is a prerequisite for the realization of endoscopic retrograde cholangiopancreatography (ERCP). When biliary cannulation is not possible, needle-knife fistulotomy (NKF) can be performed. However, when biliary access is not successfully achieved even after performing NKF, it is possible to interrupt the procedure, and repeat the ERCP after a short interval. OBJECTIVE: The aim of this study is to analyze if repeating an ERCP after a short interval of 48 hours is effective in achieving biliary access after an initial NKF was unsuccessfully performed. METHODS: A total of 1024 patients with a naive papilla, that underwent ERCP between the years of 2009-2019, were retrospectively reviewed. Difficult biliary cannulation was identified in 238 of these cases and NKF was performed. Success of biliary cannulation, NKF success at the first and second ERCPs, the associations between the type of the papilla, biliary dilatation, and overall success of NKF and adverse events rates were evaluated. RESULTS: Biliary access was initially achieved in 183 (76.8%) cases. Of the 55 (23.1%) remaining cases a second attempt was performed after 48 hours, and biliary access was successfully achieved in 46 (83.6%) of them. The overall success of NKF after the first and second ERCP, the success rate was 96.2%. Papilla located out of its normal position was related to a minor chance of success at NKF (P<0.05). CONCLUSION: We conclude that when biliary access is not achieved after the performance of a NKF, a second attempt is safe and effective and should be attempted.

RESUMO CONTEXTO: A canulação biliar de sucesso é pré-requisito para a realização da colangiopancreatografia retrógrada endoscópica (CPRE). Quando a canulação biliar não é possível, a fistulotomia com auxílio do cateter Needle-Knife (NKF) pode ser realizada. Entretanto, quando o acesso biliar não é atingido mesmo após a realização de um NKF, é possível optar-se pela interrupção do procedimento, e pela repetição da CPRE após curto intervalo de 48 horas. OBJETIVO: O objetivo desse estudo é analisar se a repetição da CPRE após um curto intervalo de 48 horas é efetivo em atingir o acesso biliar, quando um NKF foi realizado inicialmente sem sucesso. MÉTODOS: Um total de 1024 pacientes com papila virgem de tratamento, submetidos à CPRE entre os anos de 2009-2019, foram retrospectivamente analisados. Canulação biliar difícil foi identificada em 238 deles, e NKF foi então realizado. Foram avaliadas as taxas de sucesso durante a canulação biliar, assim como durante a realização de NKF na primeira e segunda CPREs. A associação entre o tipo de papila, dilatação biliar e o sucesso final na realização do NFK também foi avaliada, assim como a presença de eventos adversos associados à realização do NKF. RESULTADOS: Dentre todos os NKF realizados, acesso biliar foi inicialmente atingido em 183 (76,8%) casos. Os 55 (23,1%) casos restantes, foram submetidos a uma segunda CPRE após 48 horas e o acesso biliar foi atingido em 46 (83,6%) deles, resultando em uma taxa final de sucesso, após a primeira e segunda CPREs, de 96,2%. Papila localizada fora da sua posição habitual foi relacionada a menor chance de sucesso durante a realização de NKF (P<0,05). CONCLUSÃO: Concluiu-se que quando o acesso biliar não pode ser atingido após a realização de um NKF, uma segunda CPRE é segura, efetiva e deve ser realizada.

Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome.

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.

Chromatography-Independent Fractionation and Newly Identified Molecular Features of the Adzuki Bean (Vigna angularis Willd.) ß-vignin Protein.

Adzuki seed ß-vignin, a vicilin-like globulin, has proven to exert various health-promoting biological activities, notably in cardiovascular health. A simple scalable enrichment procedure of this protein for further nutritional and functional studies is crucial. In this study, a simplified chromatography-independent protein fractionation procedure has been optimized and described. The electrophoretic analysis showed a high degree of homogeneity of ß-vignin isolate. Furthermore, the molecular features of the purified protein were investigated. The adzuki bean ß-vignin was found to have a native size of 146 kDa, and the molecular weight determined was consistent with a trimeric structure. These were identified in two main polypeptide chains (masses of 56-54 kDa) that are glycosylated polypeptides with metal binding capacity, and one minor polypeptide chain with a mass 37 kDa, wherein these features are absent. The in vitro analysis showed a high degree of digestibility of the protein (92%) and potential anti-inflammatory capacity. The results lay the basis not only for further investigation of the health-promoting properties of the adzuki bean ß-vignin protein, but also for a possible application as nutraceutical molecule.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing.

To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNA(Pro) primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues--which are exactly complementary--for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA(Pro) reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.

Determinants of Moloney murine leukemia virus Gag-Pol and genomic RNA proportions.

UNLABELLED: The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/Gag-Pol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of Gag-Pol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA. IMPORTANCE: Viruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell-and thus dictated by the components' availabilities within the cell-or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.

An equilibrium-dependent retroviral mRNA switch regulates translational recoding.

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag-Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5-10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag-Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography.

The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ(CD)](2), 132 nt, 42.8 kDa) using a (2)H-edited NMR-spectroscopy-based approach. This approach enabled the detection of (1)H-(1)H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional (1)H-(1)H correlated and (1)H-(13)C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ(CD)](2) simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ(CD)](2) functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

How retroviruses select their genomes.

As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA genomes are selected for packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of genome packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein-RNA complexes are helping to unravel the mechanism of genome packaging and providing the first glimpses of the initial stages of retrovirus assembly.

Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus.

All retroviruses specifically package two copies of their genomes during virus assembly, a requirement for strand-transfer-mediated recombination during reverse transcription. Genomic RNA exists in virions as dimers, and the overlap of RNA elements that promote dimerization and encapsidation suggests that these processes may be coupled. Both processes are mediated by the nucleocapsid domain (NC) of the retroviral Gag polyprotein. Here we show that dimerization-induced register shifts in base pairing within the Psi-RNA packaging signal of Moloney murine leukaemia virus (MoMuLV) expose conserved UCUG elements that bind NC with high affinity (dissociation constant = 75 +/- 12 nM). These elements are base-paired and do not bind NC in the monomeric RNA. The structure of the NC complex with a 101-nucleotide 'core encapsidation' segment of the MoMuLV Psi site reveals a network of interactions that promote sequence- and structure-specific binding by NC's single CCHC zinc knuckle. Our findings support a structural RNA switch mechanism for genome encapsidation, in which protein binding sites are sequestered by base pairing in the monomeric RNA and become exposed upon dimerization to promote packaging of a diploid genome.

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus.

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.