RESUMEN
Recombination is an evolutionary process by which many pathogens generate diversity and acquire novel functions. Although a common occurrence during coronavirus replication, detection of recombination is only feasible when genetically distinct viruses contemporaneously infect the same host. Here, we identify an instance of SARS-CoV-2 superinfection, whereby an individual was infected with two distinct viral variants: Alpha (B.1.1.7) and Epsilon (B.1.429). This superinfection was first noted when an Alpha genome sequence failed to exhibit the classic S gene target failure behavior used to track this variant. Full genome sequencing from four independent extracts reveals that Alpha variant alleles comprise around 75% of the genomes, whereas the Epsilon variant alleles comprise around 20% of the sample. Further investigation reveals the presence of numerous recombinant haplotypes spanning the genome, specifically in the spike, nucleocapsid, and ORF 8 coding regions. These findings support the potential for recombination to reshape SARS-CoV-2 genetic diversity.
Asunto(s)
COVID-19 , Sobreinfección , Genoma Viral/genética , Humanos , Ciudad de Nueva York/epidemiología , Recombinación Genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
BACKGROUND: Monitoring the emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is an important public health objective. We investigated how the Gamma variant was established in New York City (NYC) in early 2021 in the presence of travel restrictions that aimed to prevent viral spread from Brazil, the country where the variant was first identified. METHODS: We performed phylogeographic analysis on 15 967 Gamma sequences sampled between 10 March and 1 May 2021, to identify geographic sources of Gamma lineages introduced into NYC. We identified locally circulating Gamma transmission clusters and inferred the timing of their establishment in NYC. RESULTS: We identified 16 phylogenetically distinct Gamma clusters established in NYC (cluster sizes ranged 2-108 genomes); most of them were introduced from Florida and Illinois and only 1 directly from Brazil. By the time the first Gamma case was reported by genomic surveillance in NYC on 10 March, the majority (57%) of circulating Gamma lineages had already been established in the city for at least 2 weeks. CONCLUSIONS: Although travel from Brazil to the United States was restricted from May 2020 through the end of the study period, this restriction did not prevent Gamma from becoming established in NYC as most introductions occurred from domestic locations.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Ciudad de Nueva York/epidemiología , COVID-19/epidemiología , FilogeniaRESUMEN
Wide-scale SARS-CoV-2 genome sequencing is critical to tracking viral evolution during the ongoing pandemic. We develop the software tool, Variant Database (VDB), for quickly examining the changing landscape of spike mutations. Using VDB, we detect an emerging lineage of SARS-CoV-2 in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage (now designated as B.1.526) are L5F, T95I, D253G, E484K or S477N, D614G, and A701V. This lineage was first sequenced in late November 2020. Phylodynamic inference confirmed the rapid growth of the B.1.526 lineage. In concert with other variants, like B.1.1.7, the rise of B.1.526 appears to have extended the duration of the second wave of COVID-19 cases in NYC in early 2021. Pseudovirus neutralization experiments demonstrated that B.1.526 spike mutations adversely affect the neutralization titer of convalescent and vaccinee plasma, supporting the public health relevance of this lineage.
Asunto(s)
COVID-19/virología , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificación , COVID-19/epidemiología , Genoma Viral , Humanos , Modelos Moleculares , Mutación , New York/epidemiología , Filogenia , SARS-CoV-2/genética , Programas Informáticos , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Wide-scale SARS-CoV-2 genome sequencing is critical to tracking viral evolution during the ongoing pandemic. Variants first detected in the United Kingdom, South Africa, and Brazil have spread to multiple countries. We developed the software tool, Variant Database (VDB), for quickly examining the changing landscape of spike mutations. Using VDB, we detected an emerging lineage of SARS-CoV-2 in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage (now designated as B.1.526) are L5F, T95I, D253G, E484K or S477N, D614G, and A701V. This lineage was first sequenced in late November 2020 when it represented <1% of sequenced coronavirus genomes that were collected in New York City (NYC). By February 2021, genomes from this lineage accounted for ~32% of 3288 sequenced genomes from NYC specimens. Phylodynamic inference confirmed the rapid growth of the B.1.526 lineage in NYC, notably the sub-clade defined by the spike mutation E484K, which has outpaced the growth of other variants in NYC. Pseudovirus neutralization experiments demonstrated that B.1.526 spike mutations adversely affect the neutralization titer of convalescent and vaccinee plasma, indicating the public health importance of this lineage.
RESUMEN
Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem-loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them.
Asunto(s)
Reparación del ADN/genética , ADN/genética , Repeticiones de Microsatélite/genética , Conformación de Ácido Nucleico , ADN/química , Daño del ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Fibroblastos , Inestabilidad Genómica/genética , Humanos , Transcripción Genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.
Asunto(s)
Guanidinas/farmacología , Guanosina/análogos & derivados , Hidantoínas/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Elongación de la Transcripción Genética/efectos de los fármacos , Daño del ADN , Reparación del ADN , Guanidinas/síntesis química , Guanidinas/química , Guanosina/síntesis química , Guanosina/química , Guanosina/farmacología , Células HeLa , Humanos , Hidantoínas/síntesis química , Hidantoínas/química , Conformación Molecular , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Transcripción Genética , ADN/metabolismo , Cartilla de ADN/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Vectores Genéticos/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Modelos Biológicos , Fenotipo , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Moldes Genéticos , Elongación de la Transcripción Genética , Proteína Fluorescente RojaRESUMEN
Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in the fly Drosophila melanogaster is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, we performed genome-wide transcriptional profiling of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, we identified 23 genes that have sex-differential transcript levels at all three time points, of which 13 encode transcription factors, a significant enrichment. We focus here on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. We show that, in female genital discs, Dsx activates lz and represses Dr and AP-2. We further show that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes we observe upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. Our time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors.