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1.
Cell ; 155(3): 594-605, 2013 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-24243017

RESUMEN

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.


Asunto(s)
Transporte Activo de Núcleo Celular , VIH-1/química , ARN Mensajero/química , ARN Viral/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células HEK293 , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Poro Nuclear/metabolismo , Conformación de Ácido Nucleico , Pliegue del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo
2.
J Virol ; 96(13): e0012222, 2022 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-35674431

RESUMEN

Effective strategies to eliminate human immunodeficiency virus type 1 (HIV-1) reservoirs are likely to require more thorough characterizations of proviruses that persist on antiretroviral therapy (ART). The rarity of infected CD4+ T-cells and related technical challenges have limited the characterization of integrated proviruses. Current approaches using next-generation sequencing can be inefficient and limited sequencing depth can make it difficult to link proviral sequences to their respective integration sites. Here, we report on an efficient method by which HIV-1 proviruses and their sites of integration are amplified and sequenced. Across five HIV-1-positive individuals on clinically effective ART, a median of 41.2% (n = 88 of 209) of amplifications yielded near-full-length proviruses and their 5'-host-virus junctions containing a median of 430 bp (range, 18 to 1,363 bp) of flanking host sequence. Unexpectedly, 29.5% (n = 26 of 88) of the sequenced proviruses had structural asymmetries between the 5' and 3' long terminal repeats (LTRs), commonly in the form of major 3' deletions. Sequence-intact proviruses were detected in 3 of 5 donors, and infected CD4+ T-cell clones were detected in 4 of 5 donors. The accuracy of the method was validated by amplifying and sequencing full-length proviruses and flanking host sequences directly from peripheral blood mononuclear cell DNA. The individual proviral sequencing assay (IPSA) described here can provide an accurate, in-depth, and longitudinal characterization of HIV-1 proviruses that persist on ART, which is important for targeting proviruses for elimination and assessing the impact of interventions designed to eradicate HIV-1. IMPORTANCE The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site. Here, we describe a novel, integrated, two-step method (individual proviral sequencing assay [IPSA]) that amplifies the host-virus junction and the full-length provirus except for the last 69 bp of the 3' long terminal repeat (LTR). Using this method, we identified the integration sites of proviruses, including those that are sequence intact and replication competent or defective. Importantly, this new method identified previously unreported asymmetries between LTRs that have implications for how proviruses are detected and quantified. The IPSA method reported is unaffected by LTR asymmetries, permitting a more accurate and comprehensive characterization of the proviral landscape.


Asunto(s)
VIH-1 , Provirus , Secuencias Repetidas Terminales , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucocitos Mononucleares/virología , Provirus/genética , Provirus/metabolismo , Secuencias Repetidas Terminales/genética
3.
Nucleic Acids Res ; 49(6): e35, 2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33406226

RESUMEN

Just as eukaryotic circular RNA (circRNA) is a product of intracellular backsplicing, custom circRNA can be synthesized in vitro using a transcription template in which transposed halves of a split group I intron flank the sequence of the RNA to be circularized. Such permuted intron-exon (PIE) constructs have been used to produce circRNA versions of ribozymes, mimics of viral RNA motifs, a streptavidin aptamer, and protein expression vectors for genetic engineering and vaccine development. One limitation of this approach is the obligatory incorporation of small RNA segments (E1 and E2) into nascent circRNA at the site of end-joining. This restriction may preclude synthesis of small circRNA therapeutics and RNA nanoparticles that are sensitive to extraneous sequence, as well as larger circRNA mimics whose sequences must precisely match those of the native species on which they are modelled. In this work, we used serial mutagenesis and in vitro selection to determine how varying E1 and E2 sequences in a thymidylate synthase (td) group I intron PIE transcription template construct affects circRNA synthesis yield. Based on our collective findings, we present guidelines for the design of custom-tailored PIE transcription templates from which synthetic circRNAs of almost any sequence may be efficiently synthesized.


Asunto(s)
ARN Circular/síntesis química , Secuencia de Bases , Exones , Humanos , Intrones , Mutagénesis , Mutación , Conformación de Ácido Nucleico , ARN Circular/química
4.
Proc Natl Acad Sci U S A ; 116(51): 25891-25899, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31776247

RESUMEN

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for "viral reconstruction" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.


Asunto(s)
VIH-1/genética , Integración Viral/genética , Replicación Viral/genética , Antirretrovirales/uso terapéutico , Secuencia de Bases , Línea Celular , ADN Viral/genética , Farmacorresistencia Viral , Infecciones por VIH/virología , Humanos , Leucocitos Mononucleares/virología , Ganglios Linfáticos/virología , Mutación , Provirus/genética , Integración Viral/fisiología
5.
Nucleic Acids Res ; 47(11): 5922-5935, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31006814

RESUMEN

Aberrant splicing in exon 11 of the LMNA gene causes the premature aging disorder Hutchinson-Gilford Progeria Syndrome. A de novo C1824T mutation activates an internal alternative 5' splice site, resulting in formation of the disease-causing progerin protein. The underlying mechanism for this 5' splice site selection is unknown. Here, we have applied a combination of targeted mutational analysis in a cell-based system and structural mapping by SHAPE-MaP to comprehensively probe the contributions of primary sequence, secondary RNA structure and linear splice site position in determining in vivo mechanisms of splice site choice in LMNA. While splice site choice is in part defined by sequence complementarity to U1 snRNA, we identify RNA secondary structural elements near the alternative 5' splice sites and show that splice site choice is significantly influenced by the structural context of the available splice sites. Furthermore, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5' splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results demonstrate that 5' splice site selection in LMNA is determined by an intricate interplay among RNA sequence, secondary structure and splice site position.


Asunto(s)
Empalme Alternativo , Lamina Tipo A/genética , ARN/química , Análisis Mutacional de ADN , Exones , Fibroblastos/metabolismo , Células HEK293 , Humanos , Lamina Tipo A/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Mutación Puntual , Progeria/genética , Estructura Secundaria de Proteína , Sitios de Empalme de ARN , Empalme del ARN , ARN Nuclear Pequeño , Síndrome
6.
Methods ; 167: 105-116, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31009771

RESUMEN

RNA is a regulator and catalyst of many cellular processes. Efforts to therapeutically harness RNA began with the discovery of myriad coding and non-coding RNAs and their versatile modes of action. However, due to its dynamic structure and the polar and repetitive nature of its surface, RNA presents a challenging target for drug design. For an RNA to be druggable, it must contain a motif that assumes a nearly fixed and unique conformation that a small molecule can recognize and bind consistently and with high affinity. Hence, reliable methods for determining the secondary and tertiary structures of RNA, and even the features and occupancy of potential drug binding sites are of utmost importance for the effective design of RNA-based therapeutics. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) has emerged as such a method, by which RNA secondary structure can be probed at single-nucleotide resolution, under a variety of conditions, and in the presence of RNA-specific small-molecule ligands. In this review, we describe an in-depth protocol for using SHAPE-MaP to characterize RNA-small molecule interactions in cell culture (in cellulo). This method can be applied to transcripts of any size or abundance, and to determine the sites and affinities of small molecule binding, making it an essential and versatile tool for drug discovery.


Asunto(s)
Conformación de Ácido Nucleico/efectos de los fármacos , ARN/química , Análisis de Secuencia de ARN/métodos , Bibliotecas de Moléculas Pequeñas/química , Acilación/efectos de los fármacos , Humanos , Ligandos , ARN/efectos de los fármacos , ARN/ultraestructura , Bibliotecas de Moléculas Pequeñas/farmacología
7.
Nucleic Acids Res ; 46(19): 10432-10447, 2018 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-30102382

RESUMEN

Long non-coding RNAs (lncRNAs) have emerged as key players in gene regulation. However, our incomplete understanding of the structure of lncRNAs has hindered molecular characterization of their function. Maternally expressed gene 3 (Meg3) lncRNA is a tumor suppressor that is downregulated in various types of cancer. Mechanistic studies have reported a role for Meg3 in epigenetic regulation by interacting with chromatin-modifying complexes such as the polycomb repressive complex 2 (PRC2), guiding them to genomic sites via DNA-RNA triplex formation. Resolving the structure of Meg3 RNA and characterizing its interactions with cellular binding partners will deepen our understanding of tumorigenesis and provide a framework for RNA-based anti-cancer therapies. Herein, we characterize the architectural landscape of Meg3 RNA and its interactions with PRC2 from a functional standpoint.


Asunto(s)
Epigénesis Genética , Neoplasias/genética , Conformación de Ácido Nucleico , ARN Largo no Codificante/genética , Cromatina/química , Cromatina/genética , ADN/química , ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Humanos , ARN Largo no Codificante/química
8.
Nucleic Acids Res ; 45(11): 6805-6821, 2017 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-28383682

RESUMEN

Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although a number nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Herein, we used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. We thus characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, we identified sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work constitutes the most extensive structural characterization of a viral lncRNA and interactions with its protein partners in discrete biological contexts, providing a broad framework for understanding the roles of PAN RNA in KSHV infection.


Asunto(s)
Herpesvirus Humano 8/genética , ARN Mensajero/metabolismo , ARN Nuclear/metabolismo , ARN Viral/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citoplasma/metabolismo , Citoplasma/virología , Herpesvirus Humano 8/metabolismo , Humanos , Secuencias Invertidas Repetidas , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Polimorfismo de Nucleótido Simple , Unión Proteica , ARN Mensajero/genética , ARN Nuclear/genética , ARN Viral/genética , Células Tumorales Cultivadas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral
10.
Nucleic Acids Res ; 43(9): 4676-86, 2015 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-25855816

RESUMEN

The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE 'conformers', we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.


Asunto(s)
VIH-1/genética , ARN Viral/química , Secuencias Reguladoras de Ácido Ribonucleico , Replicación Viral/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Fusión gag-pol/metabolismo , Genes env , VIH-1/fisiología , Mutación , Conformación de Ácido Nucleico , ARN Viral/metabolismo
11.
Retrovirology ; 13(1): 87, 2016 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-27998286

RESUMEN

BACKGROUND: Although next generation sequencing (NGS) offers the potential for studying virus populations in unprecedented depth, PCR error, amplification bias and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. Here we report a method, termed ultrasensitive Single-Genome Sequencing (uSGS), for NGS library construction and analysis that eliminates PCR errors and recombinants, and generates single-genome sequences of the same quality as the "gold-standard" of HIV-1 single-genome sequencing assay but with more than 100-fold greater depth. RESULTS: Primer ID tagged cDNA was synthesized from mixtures of cloned BH10 wild-type and mutant HIV-1 transcripts containing ten drug resistance mutations. First, the resultant cDNA was divided and NGS libraries were generated in parallel using two methods: uSGS and a method applying long PCR primers to attach the NGS adaptors (LP-PCR-1). Second, cDNA was divided and NGS libraries were generated in parallel comparing 3 methods: uSGS and 2 methods adapted from more recent reports using variations of the long PCR primers to attach the adaptors (LP-PCR-2 and LP-PCR-3). Consistently, the uSGS method amplified a greater proportion of cDNAs, averaging 30% compared to 13% for LP-PCR-1, 21% for LP-PCR-2 and 14% for LP-PCR-3. Most importantly, when the uSGS sequences were binned according to their primer IDs, 94% of the bins did not contain PCR recombinant sequences versus only 55, 75 and 65% for LP-PCR-1, 2 and 3, respectively. Finally, when uSGS was applied to plasma samples from HIV-1 infected donors, both frequent and rare variants were detected in each sample and neighbor-joining trees revealed clusters of genomes driven by the linkage of these mutations, showing the lack of PCR recombinants in the datasets. CONCLUSIONS: The uSGS assay can be used for accurate detection of rare variants and for identifying linkage of rare alleles associated with HIV-1 drug resistance. In addition, the method allows accurate in-depth analyses of the complex genetic relationships of viral populations in vivo.


Asunto(s)
Genoma Viral , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Viral/genética , Mapeo Cromosómico , Cartilla de ADN , Exactitud de los Datos , Farmacorresistencia Viral , Biblioteca de Genes , Infecciones por VIH/virología , Humanos , Mutación , Reacción en Cadena de la Polimerasa
12.
Nucleic Acids Res ; 41(9): 5075-89, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23531545

RESUMEN

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.


Asunto(s)
Virus del Dengue/genética , Genoma Viral , ARN Viral/química , Regiones no Traducidas , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Oligonucleótidos Antisentido/química
13.
Nucleic Acids Res ; 41(13): 6637-49, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23640333

RESUMEN

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem-loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.


Asunto(s)
VIH-2/genética , ARN Viral/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Regiones no Traducidas 5' , Secuencia de Bases , Ácido Edético/análogos & derivados , Ácido Edético/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Pliegue del ARN
14.
Nucleic Acids Res ; 41(6): 3874-87, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23382176

RESUMEN

A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate-a key difference between monomeric and dimeric RTs.


Asunto(s)
ADN/química , Transcriptasa Inversa del VIH/química , ARN/química , Secuencia de Aminoácidos , ADN/metabolismo , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , ARN/metabolismo , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/enzimología
15.
Nature ; 453(7192): 184-9, 2008 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-18464735

RESUMEN

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.


Asunto(s)
Replicación del ADN , ADN/biosíntesis , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH/enzimología , ARN/metabolismo , Transcripción Reversa , Sitios de Unión , Catálisis , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , VIH/genética , Hidrólisis , Ligandos , ARN/genética , Especificidad por Sustrato , Moldes Genéticos
16.
Viruses ; 16(1)2024 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-38257808

RESUMEN

The principal barrier to an HIV-1 cure is the persistence of infected cells harboring replication-competent proviruses despite antiretroviral therapy (ART). HIV-1 transcriptional suppression, referred to as viral latency, is foremost among persistence determinants, as it allows infected cells to evade the cytopathic effects of virion production and killing by cytotoxic T lymphocytes (CTL) and other immune factors. HIV-1 persistence is also governed by cellular proliferation, an innate and essential capacity of CD4+ T cells that both sustains cell populations over time and enables a robust directed response to immunological threats. However, when HIV-1 infects CD4+ T cells, this capacity for proliferation can enable surreptitious HIV-1 propagation without the deleterious effects of viral gene expression in latently infected cells. Over time on ART, the HIV-1 reservoir is shaped by both persistence determinants, with selective forces most often favoring clonally expanded infected cell populations harboring transcriptionally quiescent proviruses. Moreover, if HIV latency is incomplete or sporadically reversed in clonal infected cell populations that are replenished faster than they are depleted, such populations could both persist indefinitely and contribute to low-level persistent viremia during ART and viremic rebound if treatment is withdrawn. In this review, select genetic, epigenetic, cellular, and immunological determinants of viral transcriptional suppression and clonal expansion of HIV-1 reservoir T cells, interdependencies among these determinants, and implications for HIV-1 persistence will be presented and discussed.


Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , Linfocitos T CD4-Positivos , Linfocitos T Citotóxicos , División Celular , Células Clonales
17.
Viruses ; 14(1)2022 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-35062308

RESUMEN

COVID-19 vaccines were first administered on 15 December 2020, marking an important transition point for the spread of SARS-CoV-2 in the United States (U.S.). Prior to this point in time, the virus spread to an almost completely immunologically naïve population, whereas subsequently, vaccine-induced immune pressure and prior infections might be expected to influence viral evolution. Accordingly, we conducted a study to characterize the spread of SARS-CoV-2 in the U.S. pre-vaccination, investigate the depth and uniformity of genetic surveillance during this period, and measure and otherwise characterize changing viral genetic diversity, including by comparison with more recently emergent variants of concern (VOCs). In 2020, SARS-CoV-2 spread across the U.S. in three phases distinguishable by peaks in the numbers of infections and shifting geographical distributions. Virus was genetically sampled during this period at an overall rate of ~1.2%, though there was a substantial mismatch between case rates and genetic sampling nationwide. Viral genetic diversity tripled over this period but remained low in comparison to other widespread RNA virus pathogens, and although 54 amino acid changes were detected at frequencies exceeding 5%, linkage among them was not observed. Based on our collective observations, our analysis supports a targeted strategy for worldwide genetic surveillance as perhaps the most sensitive and efficient means of detecting new VOCs.


Asunto(s)
COVID-19/virología , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Evolución Molecular , Variación Genética , Humanos , Mutación , Filogenia , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Estados Unidos/epidemiología
18.
J Biol Chem ; 285(3): 2100-12, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19833723

RESUMEN

In this study, we identify determinants in dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) necessary for human immunodeficiency virus, type 1 (HIV-1), transmission. Although human B cell lines expressing DC-SIGN efficiently capture and transmit HIV-1 to susceptible target cells, cells expressing the related molecule liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) do not. To understand the differences between DC-SIGN and L-SIGN that affect HIV-1 interactions, we developed Raji B cell lines expressing different DC-SIGN/L-SIGN chimeras. Testing of the chimeras demonstrated that replacement of the DC-SIGN carbohydrate-recognition domain (CRD) with that of L-SIGN was sufficient to impair virus binding and prevent transmission. Conversely, the ability to bind and transmit HIV-1 was conferred to L-SIGN chimeras containing the DC-SIGN CRD. We identified Trp-258 in the DC-SIGN CRD to be essential for HIV-1 transmission. Although introduction of a K270W mutation at the same position in L-SIGN was insufficient for HIV-1 binding, an L-SIGN mutant molecule with K270W and a C-terminal DC-SIGN CRD subdomain transmitted HIV-1. These data suggest that DC-SIGN structural elements distinct from the oligosaccharide-binding site are required for HIV-1 glycoprotein selectivity.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/transmisión , VIH-1 , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Alelos , Animales , Linfocitos B/metabolismo , Moléculas de Adhesión Celular/genética , Línea Celular , Regulación de la Expresión Génica , Humanos , Lectinas/metabolismo , Lectinas Tipo C/genética , Ratones , Modelos Moleculares , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo
19.
Biochim Biophys Acta ; 1804(5): 1202-12, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-19665597

RESUMEN

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) catalyzes synthesis of integration-competent, double-stranded DNA from the single-stranded viral RNA genome, combining both polymerizing and hydrolytic functions to synthesize approximately 20,000 phosphodiester bonds. Despite a wealth of biochemical studies, the manner whereby the enzyme adopts different orientations to coordinate its DNA polymerase and ribonuclease (RNase) H activities has remained elusive. Likewise, the lower processivity of HIV-1 RT raises the issue of polymerization site targeting, should the enzyme re-engage its nucleic acid substrate several hundred nucleotides from the primer terminus. Although X-ray crystallography has clearly contributed to our understanding of RT-containing nucleoprotein complexes, it provides a static picture, revealing few details regarding motion of the enzyme on the substrate. Recent development of site-specific footprinting and the application of single molecule spectroscopy have allowed us to follow individual steps in the reverse transcription process with significantly greater precision. Progress in these areas and the implications for investigational and established inhibitors that interfere with RT motion on nucleic acid is reviewed here.


Asunto(s)
Transcriptasa Inversa del VIH/química , ADN Viral/metabolismo , Humanos , Conformación Molecular
20.
medRxiv ; 2021 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-34127980

RESUMEN

In 2020, SARS-CoV-2 spread across the United States (U.S.) in three phases distinguished by peaks in the numbers of infections and shifting geographical distribution. We investigated the viral genetic diversity in each phase using sequences publicly available prior to December 15 th , 2020, when vaccination was initiated in the U.S. In Phase 1 (winter/spring), sequences were already dominated by the D614G Spike mutation and by Phase 3 (fall), genetic diversity of the viral population had tripled and at least 54 new amino acid changes had emerged at frequencies above 5%, several of which were within known antibody epitopes. These findings highlight the need to track the evolution of SARS-CoV-2 variants in the U.S. to ensure continued efficacy of vaccines and antiviral treatments. ONE SENTENCE SUMMARY: SARS-CoV-2 genetic diversity in the U.S. increased 3-fold in 2020 and 54 emergent nonsynonymous mutations were detected.

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