RESUMO
BACKGROUND: Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. RESULTS: Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5' and 3' LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. CONCLUSIONS: XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.
Assuntos
Retrovirus Endógenos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma Viral , Fases de Leitura Aberta/genética , Sequências Repetidas Terminais/genética , Xenopus/genética , Xenopus/virologia , Animais , Retrovirus Endógenos/classificação , Evolução Molecular , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Provírus/genética , Infecções por Retroviridae/virologiaRESUMO
Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions.IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.
Assuntos
Especificidade de Hospedeiro/genética , Vírus da Leucemia Murina/classificação , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/virologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Molecular , Genoma Viral , Vírus da Leucemia Murina/genética , Camundongos , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , Homologia de Sequência , Sequências Repetidas Terminais , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ~24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ~50%. We hypothesize that the interaction between RUNX1 and CBFß is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFß interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFß directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFß interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.
Assuntos
Benzodiazepinas/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ativação Transcricional/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Western Blotting , Subunidade beta de Fator de Ligação ao Core/genética , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência/métodos , Vetores Genéticos/genética , Hematopoese/efeitos dos fármacos , Técnicas Histológicas , Humanos , Imunoprecipitação , Células Jurkat , Camundongos , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas/métodos , Ressonância de Plasmônio de Superfície , Peixe-ZebraRESUMO
CDK9/cyclin T1, a key enzyme in HIV-1 transcription, is negatively regulated by 7SK RNA and the HEXIM1 protein. Dephosphorylation of CDK9 on Thr(186) by protein phosphatase 1 (PP1) in stress-induced cells or by protein phosphatase M1A in normally growing cells activates CDK9. Our previous studies showed that HIV-1 Tat protein binds to PP1 through the Tat Q(35)VCF(38) sequence, which is similar to the PP1-binding RVXF motif and that this interaction facilitates HIV-1 transcription. In the present study, we analyzed the effect of expression of the central domain of nuclear inhibitor of PP1 (cdNIPP1) in an engineered cell line and also when cdNIPP1 was expressed as part of HIV-1 pNL4-3 in place of nef. Stable expression of cdNIPP1 increased CDK9 phosphorylation on Thr(186) and the association of CDK9 with 7SK RNA. The stable expression of cdNIPP1 disrupted the interaction of Tat and PP1 and inhibited HIV-1 transcription. Expression of cdNIPP1 as a part of the HIV-1 genome inhibited HIV-1 replication. Our study provides a proof-of-concept for the future development of PP1-targeting compounds as inhibitors of HIV-1 replication.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Endorribonucleases/fisiologia , HIV-1/genética , Fosfoproteínas Fosfatases/fisiologia , Proteínas de Ligação a RNA/fisiologia , Transcrição Gênica , Animais , Fármacos Anti-HIV , Linhagem Celular , Endorribonucleases/genética , Produtos do Gene tat/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/genética , Coelhos , Treonina/metabolismo , Replicação ViralRESUMO
APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) replication. HIV-1 synthesizes a viral infectivity factor (Vif) to counter A3G restriction. Currently, it is poorly understood how A3G expression/activity is regulated by cellular factors. Here, we show that the prolyl isomerase Pin1 protein modulates A3G expression. Pin1 was found to be an A3G-interacting protein that reduces A3G expression and its incorporation into HIV-1 virion, thereby limiting A3G-mediated restriction of HIV-1. Intriguingly, HIV-1 infection modulates the phosphorylation state of Pin1, enhancing its ability to moderate A3G activity. These new findings suggest a potential Vif-independent way for HIV-1 to moderate the cellular action of A3G.
Assuntos
Citidina Desaminase/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Peptidilprolil Isomerase/metabolismo , Replicação Viral , Desaminase APOBEC-3G , Animais , Linhagem Celular , Citidina Desaminase/genética , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírion/metabolismoRESUMO
Recent characterizations of methyl transferases as regulators of cellular processes have spurred investigations into how methylation events might influence the HIV-1 life cycle. Emerging evidence suggests that protein-methylation can positively and negatively regulate HIV-1 replication. How DNA- and RNA- methylation might impact HIV-1 is also discussed.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Metiltransferases/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Linhagem Celular , Metilação de DNA , DNA Viral/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Metilação , RNA Viral/metabolismoRESUMO
Nuclear factor-kappaB essential modulator (NEMO), also called IKKgamma, has been proposed as a 'universal' adaptor of the I-kappaB kinase (IKK) complex for stimuli such as proinflammatory cytokines, microbes, and the HTLV-I Tax oncoprotein. Currently, it remains unclear whether the many signals that activate NF-kappaB through NEMO converge identically or differently. We have adopted two approaches to answer this question. First, we generated and targeted intracellularly three NEMO-specific monoclonal antibodies (mAbs). These mAbs produced two distinct intracellular NF-kappaB inhibition profiles segregating TNFalpha from Tax activation. Second, using NEMO knockout mouse fibroblasts and 10 NEMO mutants, we found that different regions function in trans either to complement or to inhibit dominantly TNFalpha, IL-1beta, or Tax activation of NF-kappaB. For instance, NEMO (1-245 amino acids) supported Tax-mediated NF-kappaB activation, but did not serve TNFalpha- or IL-1beta signaling. Altogether, our findings indicate that while NEMO 'universally' adapts numerous NF-kappaB activators, it may do so through separable domains. We provide the first evidence that selective targeting of NEMO can abrogate oncogenic Tax signaling without affecting signals used for normal cellular metabolism.
Assuntos
Produtos do Gene tax/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Humanos , Quinase I-kappa B , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/imunologia , Alinhamento de SequênciaRESUMO
HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q(35)VCF(38), which resembles the PP1-binding "RVxF" motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the "RVxF"-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the "RVxF"-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners.
Assuntos
Fármacos Anti-HIV/farmacologia , Biocatálise/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/química , Bibliotecas de Moléculas Pequenas/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Sítios de Ligação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , HIV-1/genética , HIV-1/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Bibliotecas de Moléculas Pequenas/química , Transcrição Gênica/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Viral replication requires the use of host cell proteins and enzymes. Many viruses utilize viral helicases at various stages of their life cycle; these viruses have evolved to encode directly helicase or helicase-like proteins. In contrast, the genomes of retroviruses are devoid of viral helicases. Human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle. In this chapter, we briefly summarize the approach for assaying the RNA unwinding activity of RNA helicases measuring the effect of helicase inhibitors on HIV-1 replication.
Assuntos
RNA Helicases DEAD-box/metabolismo , HIV-1/enzimologia , Proteínas Virais/metabolismo , Antivirais/metabolismo , RNA Helicases DEAD-box/genética , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genéticaRESUMO
The majority of HIV-1-infected neonates and infants have a higher level of viremia and develop AIDS more rapidly than infected adults, including differences seen in clinical manifestations. To determine the mechanisms of HIV-1 infection in neonates vs. adults, we compared the replication kinetics of HIV-1 in neonatal (cord) and adult blood T lymphocytes and monocyte-derived macrophages (MDM) from seven different donors. We found that HIV-1 replicated 3-fold better in cord blood T lymphocytes compared with adult blood T lymphocytes and 9-fold better in cord MDM than adult MDM. We also show that this differential HIV-1 replication did not depend on differences in cell proliferative capabilities, cell surface expression of CD4, CXCR4, and CCR5, or in the amount of PCR products of reverse transcription, DNA synthesis, and translocation of preintegration complex into the nucleus in cord and adult T lymphocytes and MDM. Furthermore, using a single-cycle replication competent HIV-1-NL4-3-Env(-) luciferase amphotropic virus, which measures HIV-1 transcriptional activity independent of receptor and coreceptor expression, we found there was a 3-fold increase of HIV-1 LTR-driven luciferase expression in cord T lymphocytes compared with adult T lymphocytes and 10-fold in cord MDM than in adult MDM. The HIV-1 LTR-driven luciferase expression correlated with HIV-1 LTR transcription, as measured by ribonuclease protection assay. These data suggest that the increased replication of HIV-1 in cord blood compared with adult blood mononuclear cells is regulated at the level of HIV-1 gene expression, resulting in a higher level of viremia and faster disease progression in neonates than adults.
Assuntos
Regulação Viral da Expressão Gênica , HIV-1/fisiologia , Leucócitos Mononucleares/virologia , Replicação Viral , Adulto , Antígenos CD4/metabolismo , Proliferação de Células , Sangue Fetal/citologia , Sangue Fetal/virologia , HIV-1/genética , Humanos , Recém-Nascido , Leucócitos Mononucleares/citologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) encodes a 40-kDa Tax phosphoprotein. Tax is a transcriptional activator which modulates expression of the viral long terminal repeat and transcription of many cellular genes. Because Tax is a critical HTLV-1 factor which mediates viral transformation of T cells during the genesis of adult T-cell leukemia, it is important to understand the processes which can activate or inactivate Tax function. Here, we report that ubiquitination of Tax is a posttranscriptional mechanism which regulates Tax function. We show that ubiquitination does not target Tax for degradation by the proteasome. Rather, ubiquitin addition modifies Tax in a proteasome-independent manner from an active to a less-active transcriptional form.