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1.
Proc Natl Acad Sci U S A ; 116(21): 10504-10509, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31048506

RESUMO

To replicate in a new host, lentiviruses must adapt to exploit required host factors and evade species-specific antiviral proteins. Understanding how host protein variation drives lentivirus adaptation allowed us to expand the host range of HIV-1 to pigtail macaques. We have previously derived a viral swarm (in the blood of infected animals) that can cause AIDS in this new host. To further exploit this reagent, we generated infectious molecular clones (IMCs) from the viral swarm. We identified clones with high replicative capacity in pigtail peripheral blood mononuclear cells (PBMC) in vitro and used in vivo replication to select an individual IMC, named stHIV-A19 (for simian tropic HIV-1 clone A19), which recapitulated the phenotype obtained with the viral swarm. Adaptation of HIV-1 in macaques led to the acquisition of amino acid changes in viral proteins, such as capsid (CA), that are rarely seen in HIV-1-infected humans. Using stHIV-A19, we show that these CA changes confer a partial resistance to the host cell inhibitor Mx2 from pigtail macaques, but that complete resistance is associated with a fitness defect. Adaptation of HIV-1 to a new host will lead to a more accurate animal model and a better understanding of virus-host interactions.


Assuntos
Adaptação Biológica , Modelos Animais de Doenças , Infecções por HIV , HIV-1 , Animais , Proteínas do Capsídeo/genética , Evolução Molecular , Especificidade de Hospedeiro , Macaca nemestrina , Replicação Viral
2.
PLoS Pathog ; 14(1): e1006824, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29377940

RESUMO

The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice site(s). Overall, our data indicate complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication.


Assuntos
HIV-1/genética , Splicing de RNA/genética , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Replicação Viral/genética , Sequência de Bases , Células Cultivadas , Genoma Viral/genética , Células HEK293 , Humanos , Mutagênese/fisiologia , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Nature ; 550(7674): 124-127, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28953888

RESUMO

Vertebrate genomes exhibit marked CG suppression-that is, lower than expected numbers of 5'-CG-3' dinucleotides. This feature is likely to be due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG suppression of their hosts. This property of viral genomes is unexplained. Here we show, using synonymous mutagenesis, that CG suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, associated with cytoplasmic RNA depletion, and was exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused screen using small inhibitory RNAs revealed that zinc-finger antiviral protein (ZAP) inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG content mimicked random nucleotide sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking-immunoprecipitation-sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG suppression to identify non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defence.


Assuntos
Fosfatos de Dinucleosídeos/genética , Sequência Rica em GC/genética , HIV-1/genética , HIV-1/imunologia , RNA Viral/genética , RNA Viral/imunologia , Linhagem Celular , Citoplasma/genética , Citoplasma/virologia , HIV-1/crescimento & desenvolvimento , Humanos , Imunoprecipitação , Mutagênese , Mutação , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/genética
4.
PLoS Pathog ; 9(9): e1003667, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086139

RESUMO

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an 'assisted evolution' approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIV(mac239) Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection.


Assuntos
Proteínas de Transporte/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/genética , Linhagem Celular , Modelos Animais de Doenças , Infecções por HIV/genética , HIV-1/genética , Humanos , Macaca mulatta , Mutação , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Especificidade da Espécie , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
5.
Proc Natl Acad Sci U S A ; 107(45): 19496-501, 2010 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-20974973

RESUMO

The resurrection of endogenous retroviruses from inactive molecular fossils has allowed the investigation of interactions between extinct pathogens and their hosts that occurred millions of years ago. Two such paleoviruses, chimpanzee endogenous retrovirus-1 and -2 (CERV1 and CERV2), are relatives of modern MLVs and are found in the genomes of a variety of Old World primates, but are absent from the human genome. No extant CERV1 and -2 proviruses are known to encode functional proteins. To investigate the host range restriction of these viruses, we attempted to reconstruct functional envelopes by generating consensus genes and proteins. CERV1 and -2 enveloped MLV particles infected cell lines from a range of mammalian species. Using CERV2 Env-pseudotyped MLV reporters, we identified copper transport protein 1 (CTR1) as a receptor that was presumably used by CERV2 during its ancient exogenous replication in primates. Expression of human CTR1 was sufficient to confer CERV2 permissiveness on otherwise resistant hamster cells, and CTR1 knockdown or CuCl(2) treatment specifically inhibited CERV2 infection of human cells. Mutations in highly conserved CTR1 residues that have rendered hamster cells resistant to CERV2 include a unique deletion in a copper-binding motif. These CERV2 receptor-inactivating mutations in hamster CTR1 are accompanied by apparently compensating changes, including an increased number of extracellular copper-coordinating residues, and this may represent an evolutionary barrier to the acquisition of CERV2 resistance in primates.


Assuntos
Retrovirus Endógenos/química , Extinção Biológica , Receptores Virais/isolamento & purificação , Animais , Proteínas de Transporte de Cátions/genética , Transportador de Cobre 1 , Cricetinae , Humanos , Dados de Sequência Molecular , Pan troglodytes/virologia , Vírus/genética , Vírus/patogenicidade
6.
RNA ; 14(12): 2580-96, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18978028

RESUMO

microRNAs (miRNAs) regulate the expression of mRNAs in animals and plants through miRNA-containing ribonucleoprotein particles (RNPs). At the core of these miRNA silencing effector complexes are the Argonaute (AGO) proteins that bind miRNAs and mediate target mRNA recognition. We generated HEK293 cell lines stably expressing epitope-tagged human AGO proteins and other RNA silencing-related proteins and used these cells to purify miRNA-containing RNPs. Mass spectrometric analyses of the proteins associated with different AGO proteins revealed a common set of helicases and mRNA-binding proteins, among them the three trinucleotide repeat containing proteins 6 (TNRC6A,-B,-C). mRNA microarray analyses of these miRNA-associated RNPs revealed that AGO and TNRC6 proteins bind highly similar sets of transcripts enriched in binding sites for highly expressed endogenous miRNAs, indicating that the TNRC6 proteins are a component of the mRNA-targeting miRNA silencing complex. Together with the very similar proteomic composition of each AGO complex, this result suggests substantial functional redundancy within families of human AGO and TNRC6 proteins. Our results further demonstrate that we have developed an effective biochemical approach to identify physiologically relevant human miRNA targets.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , RNA Mensageiro/metabolismo , Linhagem Celular , Humanos , Imunoprecipitação , MicroRNAs/metabolismo , Proteoma , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismo , Transfecção
7.
PLoS Pathog ; 4(10): e1000181, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18927623

RESUMO

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5alpha and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a 'fossil record' of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5alpha proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species-dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.


Assuntos
Citidina Desaminase/fisiologia , Retrovirus Endógenos/fisiologia , Gammaretrovirus/fisiologia , Primatas/virologia , Proteínas/fisiologia , Replicação Viral , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Citidina Desaminase/genética , Evolução Molecular , Interações Hospedeiro-Patógeno/genética , Humanos , Macaca mulatta/genética , Macaca mulatta/fisiologia , Macaca mulatta/virologia , Camundongos/genética , Camundongos/fisiologia , Camundongos/virologia , Modelos Moleculares , Células NIH 3T3 , Pan troglodytes/genética , Pan troglodytes/fisiologia , Pan troglodytes/virologia , Filogenia , Primatas/genética , Proteínas/genética , Ubiquitina-Proteína Ligases , Replicação Viral/genética
8.
Genetics ; 175(1): 267-75, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17110480

RESUMO

The accumulation of slightly deleterious mutations in populations leads to the buildup of a genetic load and can cause the extinction of populations of small size. Mutation-accumulation experiments have been used to study this process in a wide variety of organisms, yet the exact mutational underpinnings of genetic loads and their fitness consequences remain poorly characterized. Here, we use an abiotic system of RNA populations evolving continuously in vitro to examine the molecular events that can instigate a genetic load. By tracking the fitness decline of ligase ribozyme populations with bottleneck sizes between 100 and 3000 molecules, we detected the appearance and subsequent fixation of both slightly deleterious mutations and advantageous mutations. Smaller populations went extinct in significantly fewer generations than did larger ones, supporting the notion of a mutational meltdown. These data suggest that mutation accumulation was an important evolutionary force in the prebiotic RNA world and that mechanisms such as recombination to ameliorate genetic loads may have been in place early in the history of life.


Assuntos
Deleção de Genes , Genética Populacional , Modelos Genéticos , Mutação , RNA Ligase (ATP)/genética , RNA Catalítico/genética , Sequência de Bases , Evolução Biológica , Deriva Genética , Carga Genética , Dados de Sequência Molecular , RNA Ligase (ATP)/química , RNA Catalítico/química , Recombinação Genética , Seleção Genética
9.
Mol Cell ; 24(5): 771-783, 2006 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-17157259

RESUMO

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases Associadas a Fase S/metabolismo , Sensibilidade e Especificidade , Análise Serial de Tecidos , Técnicas do Sistema de Duplo-Híbrido
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