RESUMO
Embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. Phenotypes associated with environmental stress suggest that some cell types are more sensitive to stress than others, but the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single-cell RNA sequencing (RNA-seq) to identify cell types and molecular programs driving phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in developmental timing. The notochord is particularly sensitive to temperature, which we map to a specialized cell type: sheath cells. These cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and anatomic defects. Our study demonstrates that whole-animal single-cell RNA-seq can identify mechanisms for developmental robustness and pinpoint cell types that constitute key failure points.
Assuntos
Proteostase , Peixe-Zebra , Animais , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Temperatura , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.
Assuntos
Mapeamento Cromossômico/métodos , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Genoma Humano , Estudo de Associação Genômica Ampla , Genômica , Humanos , Locos de Características Quantitativas , Fatores de Transcrição/genéticaRESUMO
Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Adolescente , Diferenciação Celular/genética , Feminino , Genes Homeobox/genética , Histonas/genética , Humanos , Mioblastos/fisiologia , Fatores de Transcrição/genéticaRESUMO
Current design of serological tests utilizes conservative immunoassay approaches and is focused on fast and convenient assay development, throughput, straightforward measurements, and affordability. Limitations of common serological assays include semiquantitative measurements, cross-reactivity, lack of reference standards, and no differentiation between human immunoglobulin subclasses. In this study, we suggested that a combination of immunoaffinity enrichments with targeted proteomics would enable rational design and development of serological assays of infectious diseases, such as COVID-19. Immunoprecipitation-targeted proteomic assays allowed for sensitive and specific measurements of NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum and enabled differential quantification of anti-SARS-CoV-2 antibody isotypes (IgG, IgA, IgM, IgD, and IgE) and individual subclasses (IgG1-4 and IgA1-2) in plasma and saliva. Simultaneous evaluation of the numerous antigen-antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic performance. Further validation revealed that anti-RBD IgG1, IgG3, IgM, and IgA1 levels were significantly elevated in convalescent plasma, while IgG2, IgG4, and IgA2 were not informative. Anti-RBD IgG1 levels in convalescent (2138 ng/mL) vs negative (95 ng/mL) plasma revealed 385 ng/mL as a cutoff to detect COVID-19 convalescent plasma. Immunoprecipitation-targeted proteomic assays will facilitate improvement and standardization of the existing serological tests, enable rational design of novel tests, and offer tools for the comprehensive investigation of immunoglobulin subclass cooperation in immune response.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , COVID-19/diagnóstico , COVID-19/terapia , Teste para COVID-19 , Humanos , Imunização Passiva , Imunoglobulina A , Imunoglobulina D , Imunoglobulina E , Imunoglobulina G , Imunoglobulina M , Imunoprecipitação , Proteômica , Soroterapia para COVID-19RESUMO
The aim of this study was to quantify the effect of iodinated contrast media (ICM) conservation measures implemented at a single health system during a global shortage, comparing the 12-month period before intervention and the 14-day period after intervention. The mean daily utilization of contrast-enhanced CT decreased from 112 to 44 examinations, the mean ICM volume per CECT examination decreased from 88 to 74 mL, and the mean daily ICM use decreased from 9.9 to 3.3 L.
Assuntos
Meios de Contraste , Compostos de Iodo , Humanos , Meios de Contraste/efeitos adversos , Fatores de RiscoRESUMO
Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to â¼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Análise de Célula Única/métodos , Sistemas CRISPR-Cas , Lentivirus/genética , RNA Guia de Cinetoplastídeos/genética , Análise de Sequência de RNARESUMO
Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single-cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.
Assuntos
Células/citologia , Microscopia de Fluorescência/instrumentação , Linhagem Celular , Forma do Núcleo Celular/efeitos dos fármacos , Citometria de Fluxo , Testes Genéticos , Humanos , Sinais de Localização Nuclear/metabolismo , Paclitaxel/farmacologia , Fenótipo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
BACKGROUND: Hyperphosphatemia is associated with increased fibroblast growth factor 23 (FGF23), arterial calcification, and cardiovascular mortality. Effects of phosphate-lowering medication on vascular calcification and arterial stiffness in CKD remain uncertain. METHODS: To assess the effects of non-calcium-based phosphate binders on intermediate cardiovascular markers, we conducted a multicenter, double-blind trial, randomizing 278 participants with stage 3b or 4 CKD and serum phosphate >1.00 mmol/L (3.10 mg/dl) to 500 mg lanthanum carbonate or matched placebo thrice daily for 96 weeks. We analyzed the primary outcome, carotid-femoral pulse wave velocity, using a linear mixed effects model for repeated measures. Secondary outcomes included abdominal aortic calcification and serum and urine markers of mineral metabolism. RESULTS: A total of 138 participants received lanthanum and 140 received placebo (mean age 63.1 years; 69% male, 64% White). Mean eGFR was 26.6 ml/min per 1.73 m2; 45% of participants had diabetes and 32% had cardiovascular disease. Mean serum phosphate was 1.25 mmol/L (3.87 mg/dl), mean pulse wave velocity was 10.8 m/s, and 81.3% had abdominal aortic calcification at baseline. At 96 weeks, pulse wave velocity did not differ significantly between groups, nor did abdominal aortic calcification, serum phosphate, parathyroid hormone, FGF23, and 24-hour urinary phosphate. Serious adverse events occurred in 63 (46%) participants prescribed lanthanum and 66 (47%) prescribed placebo. Although recruitment to target was not achieved, additional analysis suggested this was unlikely to have significantly affected the principle findings. CONCLUSIONS: In patients with stage 3b/4 CKD, treatment with lanthanum over 96 weeks did not affect arterial stiffness or aortic calcification compared with placebo. These findings do not support the role of intestinal phosphate binders to reduce cardiovascular risk in patients with CKD who have normophosphatemia. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Australian Clinical Trials Registry, ACTRN12610000650099.
Assuntos
Hiperfosfatemia/sangue , Lantânio/uso terapêutico , Fosfatos/sangue , Insuficiência Renal Crônica/sangue , Calcificação Vascular/diagnóstico por imagem , Idoso , Aorta Abdominal , Método Duplo-Cego , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Taxa de Filtração Glomerular , Humanos , Hiperfosfatemia/tratamento farmacológico , Hiperfosfatemia/etiologia , Lantânio/efeitos adversos , Masculino , Pessoa de Meia-Idade , Hormônio Paratireóideo/sangue , Fosfatos/urina , Análise de Onda de Pulso , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
Asymptomatic urolithiasis is common and of mixed composition in patients with ß-thalassaemia major. Twenty-seven subjects were imaged using dual-energy computer tomography to determine the presence and composition of urolithiasis. The prevalence of urolithiasis was 59% and affected patients generally had multiple stones, often with more than one component: struvite (33%), calcium oxalate (31%) and cystine (22%). Hypercalciuria was present in 78% of subjects and calcium-containing urolithiasis was associated with reduced femoral neck Z scores.
Assuntos
Densidade Óssea/fisiologia , Hipercalcemia/epidemiologia , Urolitíase/epidemiologia , Talassemia beta/epidemiologia , Adulto , Feminino , Humanos , Hipercalcemia/diagnóstico por imagem , Hipercalcemia/metabolismo , Masculino , Pessoa de Meia-Idade , Prevalência , Urolitíase/diagnóstico por imagem , Urolitíase/metabolismo , Adulto Jovem , Talassemia beta/diagnóstico por imagem , Talassemia beta/metabolismoRESUMO
The purpose of this study was to assess the efficacy of model-based iterative reconstruction (MBIR), statistical iterative reconstruction (SIR), and filtered back projection (FBP) image reconstruction algorithms in the delineation of ureters and overall image quality on non-enhanced computed tomography of the renal tracts (NECT-KUB). This was a prospective study of 40 adult patients who underwent NECT-KUB for investigation of ureteric colic. Images were reconstructed using FBP, SIR, and MBIR techniques and individually and randomly assessed by two blinded radiologists. Parameters measured were overall image quality, presence of ureteric calculus, presence of hydronephrosis or hydroureters, image quality of each ureteric segment, total length of ureters unable to be visualized, attenuation values of image noise, and retroperitoneal fat content for each patient. There were no diagnostic discrepancies between image reconstruction modalities for urolithiasis. Overall image qualities and for each ureteric segment were superior using MBIR (67.5 % rated as 'Good to Excellent' vs. 25 % in SIR and 2.5 % in FBP). The lengths of non-visualized ureteric segments were shortest using MBIR (55.0 % measured 'less than 5 cm' vs. ASIR 33.8 % and FBP 10 %). MBIR was able to reduce overall image noise by up to 49.36 % over SIR and 71.02 % over FBP. MBIR technique improves overall image quality and visualization of ureters over FBP and SIR.
Assuntos
Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , Cálculos Ureterais/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Simian foamy viruses (SVF) are ubiquitous in nonhuman primates (NHP). SFV can be zoonotically transmitted to humans who either work with or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, a PCR assay failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.
Assuntos
Infecções por Retroviridae/epidemiologia , Vírus Espumoso dos Símios/isolamento & purificação , Migrantes , Zoonoses/epidemiologia , Animais , Bangladesh/epidemiologia , Feminino , Humanos , Macaca , Masculino , Reação em Cadeia da Polimerase , RNA Viral/sangue , Vírus Espumoso dos Símios/genéticaRESUMO
Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a ß-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence.
Assuntos
Doenças dos Macacos/virologia , Infecções por Retroviridae/veterinária , Infecções por Retroviridae/virologia , Vírus Espumoso dos Símios/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Humanos , Macaca mulatta , Dados de Sequência Molecular , Platirrinos , Vírus Espumoso dos Símios/genética , Vírus Espumoso dos Símios/isolamento & purificação , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
Foamy viruses are retroviruses whose Pol protein is synthesized without Gag from a spliced mRNA. Unlike orthoretroviruses, reverse transcription occurs during viral assembly, leading to DNA-containing virions. When prototype foamy virus Pol is expressed as an orthoretroviral-like Gag-Pol fusion protein, reverse transcription also occurs late in viral replication, as measured by the timing of reverse transcriptase sensitivity to the inhibitor 3'-azido-3'deoxythymidine (AZT). Thus, timing of reverse transcription is intrinsic to Pol itself.
Assuntos
Proteínas de Fusão gag-pol/biossíntese , Proteínas de Fusão gag-pol/genética , Regulação Viral da Expressão Gênica , Transcrição Reversa , Spumavirus/genética , Linhagem Celular , Humanos , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Spumavirus/fisiologia , Montagem de Vírus , Zidovudina/metabolismoRESUMO
Mutations in the DMD gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2-4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.
RESUMO
Mutations in the DMD gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder that manifests itself as young boys acquire motor functions. DMD is typically diagnosed at 2 to 4 years of age, but the absence of dystrophin negatively impacts muscle structure and function before overt symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Here, dystrophin deficiency was linked to marked dysregulations of cell junction protein families involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.
RESUMO
Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Transdução de Sinais , Receptores Proteína Tirosina Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Genômica , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/uso terapêuticoRESUMO
Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex and find epigenetic signatures of variation in ATAC-Seq data in this discovery cohort. We then scale up our single-cell RNA-Seq analysis by combining our data with five recently published single nucleus RNA-Seq datasets of healthy adult hearts. We find variation such as metabolic alterations by sex and immune changes by age in differential expression tests, as well as alterations in abundance of cardiomyocytes by sex and neurons with age. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs. Our analysis represents the largest single-cell analysis of cardiac variation by age and sex to date and provides a resource for further study of healthy cardiac variation and transcriptional regulation at single-cell resolution.
Assuntos
Cromatina , Análise de Célula Única , Humanos , Análise de Célula Única/métodos , Feminino , Masculino , Adulto , Cromatina/metabolismo , Cromatina/genética , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miocárdio/citologia , Caracteres Sexuais , Idoso , Fatores Etários , Envelhecimento/genética , Fatores Sexuais , Adulto Jovem , Miócitos Cardíacos/metabolismo , Coração/crescimento & desenvolvimentoRESUMO
Foamy viruses (FV) synthesize Pol from a spliced pol mRNA independently of Gag, unlike orthoretroviruses, which synthesize Pol as a Gag-Pol protein that coassembles with Gag. We found that prototype FV (PFV) mutants expressing Gag and Pol only as a Gag-Pol protein without the spliced Pol contain protease activity equivalent to that of wild-type (WT) Pol. Regardless of the presence or absence of the spliced Pol, the PFV Gag-Pol proteins can assemble into virus-like particles (VLPs), in contrast to the orthoretroviral Gag-Pol proteins, which cannot form VLPs. However, the PFV Gag-Pol VLPs have aberrant morphologies and are not infectious. In the absence of the spliced Pol, coexpression of a PFV Gag-Pol protein with Gag can produce infectious virions. Our results suggest that enzymes encoded by PFV pol (protease, reverse transcriptase, and integrase) are enzymatically active if they are synthesized as part of a Gag-Pol protein.
Assuntos
Produtos do Gene gag/metabolismo , Produtos do Gene pol/metabolismo , Spumavirus/enzimologia , Spumavirus/patogenicidade , Animais , Expressão Gênica , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Humanos , Splicing de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spumavirus/genética , Vírion/patogenicidade , Vírion/ultraestrutura , Virossomos/metabolismo , Virossomos/ultraestruturaRESUMO
Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging.