RESUMO
In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Transporte Ativo do Núcleo Celular , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , HIV-1/genética , Vírion/metabolismo , Replicação Viral , Desenvelopamento do Vírus , Integração ViralRESUMO
HIV-1 integration favors recurrent integration gene (RIG) targets and genic proviruses can confer cell survival in vivo. However, the relationship between initial RIG integrants and how these evolve in patients over time are unknown. To address these shortcomings, we built phenomenological models of random integration in silico, which were used to identify 3718 RIGs as well as 2150 recurrent avoided genes from 1.7 million integration sites across 10 in vitro datasets. Despite RIGs comprising only 13% of human genes, they harbored 70% of genic HIV-1 integrations across in vitro and patient-derived datasets. Although previously reported to associate with super-enhancers, RIGs tracked more strongly with speckle-associated domains. While depletion of the integrase cofactor LEDGF/p75 significantly reduced recurrent HIV-1 integration in vitro, LEDGF/p75 primarily occupied non-speckle-associated regions of chromatin, suggesting a previously unappreciated dynamic aspect of LEDGF/p75 functionality in HIV-1 integration targeting. Finally, we identified only six genes from patient samples-BACH2, STAT5B, MKL1, MKL2, IL2RB and MDC1-that displayed enriched integration targeting frequencies and harbored proviruses that likely contributed to cell survival. Thus, despite the known preference of HIV-1 to target cancer-related genes for integration, we conclude that genic proviruses play a limited role to directly affect cell proliferation in vivo.
Assuntos
Genômica/métodos , HIV-1/genética , Integração Viral , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células HEK293 , Infecções por HIV/genética , Humanos , Células Jurkat , Modelos Biológicos , Provírus , Fatores de Transcrição/fisiologiaRESUMO
Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor 5 (CPSF5) and serine/arginine-like protein CPSF6, regulates alternative polyadenylation (APA). Loss of CFIm function results in proximal polyadenylation site usage, shortening mRNA 3' untranslated regions (UTRs). Although CPSF6 plays additional roles in human disease, its nuclear translocation mechanism remains unresolved. Two ß-karyopherins, transportin (TNPO) 1 and TNPO3, can bind CPSF6 in vitro, and we demonstrate here that while the TNPO1 binding site is dispensable for CPSF6 nuclear import, the arginine/serine (RS)-like domain (RSLD) that mediates TNPO3 binding is critical. The crystal structure of the RSLD-TNPO3 complex revealed potential CPSF6 interaction residues, which were confirmed to mediate TNPO3 binding and CPSF6 nuclear import. Both binding and nuclear import were independent of RSLD phosphorylation, though a hyperphosphorylated mimetic mutant failed to bind TNPO3 and mislocalized to the cell cytoplasm. Although hypophosphorylated CPSF6 largely supported normal polyadenylation site usage, a significant number of mRNAs harbored unnaturally extended 3' UTRs, similar to what is observed when other APA regulators, such as CFIIm component proteins, are depleted. Our results clarify the mechanism of CPSF6 nuclear import and highlight differential roles for RSLD phosphorylation in nuclear translocation versus regulation of APA.
Assuntos
Poliadenilação/genética , Conformação Proteica , Proteínas de Ligação a RNA/química , beta Carioferinas/química , Transporte Ativo do Núcleo Celular/genética , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Fosforilação/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , RNA Mensageiro , Proteínas de Ligação a RNA/genética , beta Carioferinas/genética , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins. The accurate and efficient cleavage of Gag is essential for virion infectivity; inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by â¼95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectably affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions.IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein results in a strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that the membrane-binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.
Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Infecções por HIV/virologia , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/metabolismo , Integração Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/genética , Núcleo Celular/virologia , Células HEK293 , HIV-1/fisiologia , Humanos , Nucleocapsídeo , Transcrição Reversa , Proteínas Virais de Fusão/genética , Proteínas da Matriz Viral/genética , Vírion , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.
Assuntos
Marcação de Genes , Integrase de HIV/fisiologia , HIV-1/fisiologia , Integração Viral/fisiologia , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Internalização do Vírus , Latência Viral/fisiologiaRESUMO
The early stages of HIV-1 infection include the trafficking of the viral core into the nucleus of infected cells. However, much remains to be understood about how HIV-1 accomplishes nuclear import and the consequences of the import pathways utilized on nuclear events. The host factor cleavage and polyadenylation specificity factor 6 (CPSF6) assists HIV-1 nuclear localization and post-entry integration targeting. Here, we used a CPSF6 truncation mutant lacking a functional nuclear localization signal (NLS), CPSF6-358, and appended heterologous NLSs to rescue nuclear localization. We show that some, but not all, NLSs drive CPSF6-358 into the nucleus. Interestingly, we found that some nuclear localized CPSF6-NLS chimeras supported inefficient HIV-1 infection. We found that HIV-1 still enters the nucleus in these cell lines but fails to traffic to speckle-associated domains (SPADs). Additionally, we show that HIV-1 fails to efficiently integrate in these cell lines. Collectively, our results demonstrate that the NLS of CPSF6 facilitates steps of HIV-1 infection subsequent to nuclear import and additionally identify the ability of canonical NLS sequences to influence cargo localization in the nucleus following nuclear import.
RESUMO
Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration. However, disruption of CypA binding to the mutant capsid restored nuclear entry, integration, and infection in a CPSF6-dependent manner. Furthermore, relocalization of CypA expression from the cell cytoplasm to the nucleus failed to restore mutant HIV-1 infection. Our results clarify that sequential binding of CypA and CPSF6 to HIV-1 capsid is required for optimal nuclear entry and integration targeting, informing antiretroviral therapies that contain lenacapavir.
RESUMO
HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase with host chromatin factor LEDGF. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this role is inconsistent with LEDGF-mediated integration across gene bodies and with data indicating LEDGF is a histone chaperone that promotes transcriptional elongation. We found LEDGF is enriched in pronounced peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. Our genome-wide chromatin mapping revealed that MLL1 had a dominant role in recruiting LEDGF to promoters and the presence of LEDGF recruits RNA Pol II. Enrichment of LEDGF at TSSs correlates strongly with levels of integration across the transcribed sequences, indicating that LEDGF at TSSs contributed to integration across gene bodies. Although the N-terminal Pro-Trp-Trp-Pro (PWWP) domain of LEDGF interacts with nucleosomes containing H3K36me3, a modification thought to recruit LEDGF to chromatin, we found H3K36me3 does not contribute to gene specificity of integration. These data support a dual role model of LEDGF where it is tethered to promoters by MLL1 and recruits RNA Pol II. Subsequently, LEDGF travels across genes to effect HIV-1 integration. Our data also provides a mechanistic context for the contribution made by LEDGF to MLL1-based infant acute leukemia and acute myeloid leukemia in adults.
RESUMO
Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short phenylalanine-glycine (FG) containing peptides with isolated capsid hexamers have been characterized, how these cellular factors functionally engage with biologically relevant mature HIV-1 capsid lattices is unknown. Here we show that prion-like low complexity regions (LCRs) enable avid CPSF6, NUP153 and SEC24C binding to capsid lattices. Structural studies revealed that multivalent CPSF6 assembly is mediated by LCR-LCR interactions, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. In infected cells, avid CPSF6 LCR-mediated binding to HIV-1 cores is essential for functional virus-host interactions. The investigational drug lenacapavir accesses unoccupied hydrophobic pockets in the complex to potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results establish previously undescribed mechanisms of virus-host interactions and antiviral action.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , HIV-1 , Príons , Humanos , Proteínas do Capsídeo/metabolismo , Drogas em Investigação , Glicina/metabolismo , HIV-1/metabolismo , Interações entre Hospedeiro e Microrganismos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/metabolismo , Príons/metabolismo , Integração ViralRESUMO
A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.
Assuntos
DNA Viral , Integrases , Animais , Humanos , Domínio Catalítico , DNA Viral/metabolismo , Integrases/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Modelos Moleculares , Retroviridae/genética , Ovinos/genética , Integração ViralRESUMO
Ordinarily, RIP-induced erg-3 mutant Neurospora crassa ascospores and their erg(+) siblings do not differ in stability during long-term storage. Consequently, the frequency of RIP-induced erg-3 mutants remains about constant regardless of the time that has elapsed between ascospore harvest and germination. We found, however, that RIP-induced erg-3 mutants were apparently selectively lost with time from among the ascospores stored from a cross with the wild-isolated Carrefour Mme. Gras strain from Haiti. The Haitian strain was also found to exert a dominant suppression of meiotic silencing by unpaired DNA. Similar loss of RIP-induced erg-3 mutant ascospores was seen among the stored ascospores from a subset of crosses heterozygous for the semi-dominant Sad-1 or Sad-2 suppressors of meiotic silencing. Our results suggest that crosses suppressed in meiotic silencing can compromise the stability during storage of ascospores that inherit RIP-induced mutations.
Assuntos
DNA Fúngico/genética , Meiose , Neurospora crassa/genética , Esporos Fúngicos/citologia , Cruzamentos Genéticos , Genes Fúngicos Tipo Acasalamento , Mutação , Neurospora crassa/citologia , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/isolamento & purificação , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Studies on Neurospora chromosome segment duplications (Dps) performed since the publication of Perkins's comprehensive review in 1997 form the focus of this article. We present a brief summary of Perkins's seminal work on chromosome rearrangements, specifically, the identification of insertional and quasiterminal translocations that can segregate Dp progeny when crossed with normal sequence strains (i.e., T x N). We describe the genome defense process called meiotic silencing by unpaired DNA that renders Dp-heterozygous crosses (i.e., Dp x N) barren, which provides a basis for identifying Dps, and discuss whether other processes also might contribute to the barren phenotype of Dp x N and Dp x Dp crosses. We then turn to studies suggesting that large Dps (i.e., >300 kbp) can allow smaller gene-sized duplications to escape another genome defense process called repeat-induced point mutation (RIP), possibly by titration of the RIP machinery. Finally, we assess whether in natural populations dominant RIP suppressor Dps provide an "RIP-free" niche for evolution of new genes following the duplication of existing genes.
Assuntos
Cromossomos Fúngicos/genética , Cruzamentos Genéticos , Duplicação Gênica , Neurospora crassa/genética , Heterozigoto , Meiose , FenótipoRESUMO
The HIV-1 latent reservoir is the major barrier to an HIV cure. Due to low levels or lack of transcriptional activity, HIV-1 latent proviruses in vivo are not easily detectable and cannot be targeted by either natural immune mechanisms or molecular therapies based on protein expression. To target the latent reservoir, further understanding of HIV-1 proviral transcription is required. In this study, we demonstrate a novel role for cleavage and polyadenylation specificity factor 6 (CPSF6) in HIV-1 transcription. We show that knockout of CPSF6 hinders reactivation of latent HIV-1 proviruses by PMA in primary CD4+ cells. CPSF6 knockout reduced HIV-1 transcription, concomitant with a drastic reduction in the phosphorylation levels of Pol II and CDK9. Knockout of CPSF6 led to abnormal stabilization of protein phosphatase 2A (PP2A) subunit A, which then acted to dephosphorylate CDK9, downmodulating CDK9's ability to phosphorylate the Pol II carboxy-terminal domain. In agreement with this mechanism, incubation with the PP2A inhibitor, LB100, restored HIV-1 transcription in the CPSF6 knockout cells. Destabilization of PP2A subunit A occurs in the ubiquitin proteasome pathway, wherein CPSF6 acts as a substrate adaptor for the ITCH ubiquitin ligase. Our observations reveal a novel role of CPSF6 in HIV-1 transcription, which appears to be independent of its known roles in cleavage and polyadenylation and the targeting of preintegration complexes to the chromatin for viral DNA integration. IMPORTANCE CPSF6 is a cellular factor that regulates cleavage and polyadenylation of mRNAs and participates in HIV-1 infection by facilitating targeting of preintegration complexes to the chromatin. Our observations reveal a second role of CPSF6 in the HIV-1 life cycle that involves regulation of viral transcription through controlling the stability of protein phosphatase 2A, which in turn regulates the phosphorylation/dephosphorylation status of critical residues in CDK9 and Pol II.
Assuntos
HIV-1/genética , HIV-1/metabolismo , Latência Viral/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Reservatórios de Doenças/virologia , Células HEK293 , Humanos , Monócitos/virologia , Fosforilação , Provírus/genética , Provírus/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Integração Viral , Latência Viral/fisiologia , Replicação ViralRESUMO
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20152-w.
RESUMO
The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.
Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de Fluorescência , Virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs.IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.
Assuntos
Lentivirus/genética , Primatas/genética , Integração Viral/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Gatos/genética , Gatos/virologia , Bovinos/genética , Bovinos/virologia , Linhagem Celular , Evolução Molecular , Células HEK293 , Cavalos/genética , Cavalos/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Jurkat , Macaca mulatta/genética , Macaca mulatta/virologia , Camundongos/genética , Camundongos/virologia , Primatas/virologia , Replicação ViralRESUMO
The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.
Assuntos
Fármacos Anti-HIV , Capsídeo , HIV-1 , Humanos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Capsídeo/química , Capsídeo/efeitos dos fármacos , Microscopia Crioeletrônica , Cristalografia por Raios X , Medição da Troca de Deutério , Células HEK293 , Células HeLa , HIV-1/química , HIV-1/efeitos dos fármacos , Fatores de Poliadenilação e Clivagem de mRNA/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Domínios Proteicos , Integração ViralRESUMO
In Neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (Dp) of the translocated chromosome segment. Here, 30 breakpoint junction sequences of 12 Dp-generating translocations were determined. The breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. Insertion of sequences from LG III into LG I as translocation T(UK8-18) disrupts the eat-3 gene, which is the ortholog of the Podospora anserine gene ami1. Since ami1-homozygous Podospora crosses were reported to increase the frequency of repeat-induced point mutation (RIP), we performed crosses homozygous for a deficiency in eat-3 to test for a corresponding increase in RIP frequency. However, our results suggested that, unlike in Podospora, the eat-3 gene might be essential for ascus development in Neurospora. Duplication-heterozygous crosses are generally barren in Neurospora; however, by using molecular probes developed in this study, we could identify Dp segregants from two different translocation-heterozygous crosses, and using these we found that the barren phenotype of at least some duplication-heterozygous crosses was incompletely penetrant.
Assuntos
Duplicação Cromossômica , Cromossomos Fúngicos/genética , Genes Fúngicos , Neurospora crassa/genética , Fases de Leitura Aberta , Translocação Genética , Pontos de Quebra do Cromossomo , Deleção de Genes , Marcadores Genéticos , Hibridização Genética , FenótipoRESUMO
Repeat-induced point mutation (RIP) is a hypermutational process that alters duplicated DNA sequences in Neurospora crassa. In previous studies, five of six large ( > 100 kb) chromosome segment duplications (Dp's) examined were shown to dominantly suppress RIP in smaller (< 5 kb) duplications. The suppressor duplications were > 270 kb, whereas the lone non-suppressor duplication was approximately 117 kb. We have now screened another 33 duplications and found 29 more suppressors and four more non-suppressors. All 22 suppressor duplications whose size could be estimated were > 270 kb, whereas two newly identified non-suppressor duplications examined were 140-154 kb. RIP was suppressed in a subset of crosses heterozygous for more than one ordinarily non-suppressor duplication. These results strengthen the hypothesis that large duplications titrate out the RIP machinery and suggest the "equivalence point" for the titration is close to 300 kb.
Assuntos
Cromossomos Fúngicos/genética , Duplicação Gênica , Neurospora crassa/genética , Mutação Puntual , Cruzamentos Genéticos , DNA Fúngico/genética , Genes Dominantes , Genes Fúngicos , Heterozigoto , FenótipoRESUMO
In Neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (Dp) of the translocated chromosome segment. Here, 30 breakpoint junction sequences of 12 Dp-generating translocations were determined. The breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. Insertion of sequences from LG III into LG I as translocation T(UK8- 18) disrupts the eat-3 gene, which is the ortholog of the Podospora anserine gene ami1. Since ami1-homozygous Podospora crosses were reported to increase the frequency of repeat-induced point mutation (RIP), we performed crosses homozygous for a defi ciency in eat-3 to test for a corresponding increase in RIP frequency. However, our results suggested that, unlike in Podospora, the eat-3 gene might be essential for ascus development in Neurospora. Duplication–heterozygous crosses are generally barren in Neurospora; however, by using molecular probes developed in this study, we could identify Dp segregants from two different translocation–heterozygous crosses, and using these we found that the barren phenotype of at least some duplication–heterozygous crosses was incompletely penetrant.