RESUMO
The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/ultraestrutura , Vírion/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , HumanosRESUMO
The early events in the retrovirus assembly pathway, particularly the timing and nature of Gag translocation from the site of protein translation to the inner leaflet of the plasma membrane, are poorly understood. We have investigated the interrelationship between cytoplasmic Gag concentration and plasma membrane association using complementary live-cell biophysical fluorescence techniques in real time with both human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) Gag proteins. In particular, dual-color, z-scan fluorescence fluctuation spectroscopy in conjunction with total internal reflection fluorescence and conventional, epi-illumination imaging were utilized. Our results demonstrate that HTLV-1 Gag is capable of membrane targeting and particle assembly at low (i.e., nanomolar) cytoplasmic concentrations and that there is a critical threshold concentration (approaching micromolar) prior to the observation of HIV-1 Gag associated with the plasma membrane. These observations imply fundamental differences between HIV-1 and HTLV-1 Gag trafficking and membrane association.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Produtos do Gene gag/metabolismo , Retroviridae/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismoRESUMO
Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.
Assuntos
Antígenos HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Infecções por HTLV-II/virologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Antígenos HIV/química , Antígenos HIV/genética , HIV-1/química , HIV-1/genética , Vírus Linfotrópico T Tipo 2 Humano/química , Vírus Linfotrópico T Tipo 2 Humano/genética , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Ligação Proteica , Estrutura Secundária de Proteína , RNA Viral/genética , Alinhamento de Sequência , Especificidade da Espécie , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) has a reputation for being extremely difficult to study in cell culture. The challenges in propagating HTLV-1 has prevented a rigorous analysis of how these viruses replicate in cells, including the detailed steps involved in virus assembly. The details for how retrovirus particle assembly occurs are poorly understood, even for other more tractable retroviral systems. Recent studies on HTLV-1 using state-of-the-art cryo-electron microscopy and fluorescence-based biophysical approaches explored questions related to HTLV-1 particle size, Gag stoichiometry in virions, and Gag-Gag interactions in living cells. These results provided new and exciting insights into fundamental aspects of HTLV-1 particle assembly-which are distinct from those of other retroviruses, including HIV-1. The application of these and other novel biophysical approaches promise to provide exciting new insights into HTLV-1 replication.
Assuntos
Produtos do Gene gag/metabolismo , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírion/química , Vírion/fisiologia , Montagem de Vírus , Animais , Produtos do Gene gag/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Vírion/genéticaRESUMO
Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.
Assuntos
Citoplasma/metabolismo , HIV-1 , Vírus Linfotrópico T Tipo 1 Humano , Espectrometria de Fluorescência/métodos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Membrana Celular/metabolismo , Cor , Células HeLa , Humanos , Ácidos Mirísticos , Ligação Proteica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/químicaRESUMO
BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1) is an important human retrovirus that is a cause of adult T-cell leukemia/lymphoma. While an important human pathogen, the details regarding virus replication cycle, including the nature of HTLV-1 particles, remain largely unknown due to the difficulties in propagating the virus in tissue culture. In this study, we created a codon-optimized HTLV-1 Gag fused to an EYFP reporter as a model system to quantitatively analyze HTLV-1 particles released from producer cells. RESULTS: The codon-optimized Gag led to a dramatic and highly robust level of Gag expression as well as virus-like particle (VLP) production. The robust level of particle production overcomes previous technical difficulties with authentic particles and allowed for detailed analysis of particle architecture using two novel methodologies. We quantitatively measured the diameter and morphology of HTLV-1 VLPs in their native, hydrated state using cryo-transmission electron microscopy (cryo-TEM). Furthermore, we were able to determine HTLV-1 Gag stoichiometry as well as particle size with the novel biophysical technique of fluorescence fluctuation spectroscopy (FFS). The average HTLV-1 particle diameter determined by cryo-TEM and FFS was 71 ± 20 nm and 75 ± 4 nm, respectively. These values are significantly smaller than previous estimates made of HTLV-1 particles by negative staining TEM. Furthermore, cryo-TEM reveals that the majority of HTLV-1 VLPs lacks an ordered structure of the Gag lattice, suggesting that the HTLV-1 Gag shell is very likely to be organized differently compared to that observed with HIV-1 Gag in immature particles. This conclusion is supported by our observation that the average copy number of HTLV-1 Gag per particle is estimated to be 510 based on FFS, which is significantly lower than that found for HIV-1 immature virions. CONCLUSIONS: In summary, our studies represent the first quantitative biophysical analysis of HTLV-1-like particles and reveal novel insights into particle morphology and Gag stochiometry.
Assuntos
Produtos do Gene gag/análise , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Vírus Linfotrópico T Tipo 1 Humano/ultraestrutura , Vírion/ultraestrutura , Proteínas de Bactérias/genética , Linhagem Celular , Códon/genética , Microscopia Crioeletrônica , Produtos do Gene gag/genética , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise Espectral/métodosRESUMO
Clinical observations have revealed a strong correlation between loss of bone density in HIV-infected individuals, particularly in conjunction with the antiretroviral drug tenofovir, a nucleotide analog that inhibits HIV reverse transcriptase. The most compelling correlations have been observed in clinical studies involving young children and adolescents. These observations strongly suggest that bone density is being affected during active bone growth and development, implicating a role for tenofovir in bone loss. Here we discuss the literature and potential mechanisms for how tenofovir-associated bone loss may arise, which likely involves perturbation of cellular DNA synthesis and gene expression. Elucidation of the mechanism(s) involved in tenofovir-mediated bone loss will help in developing adjuvant therapies to reduce tenofovir-associated bone density loss.
RESUMO
There is strong clinical evidence that implicates tenofovir in the loss of bone mineral density during treatment of human immunodeficiency virus infection. In this study, we sought to test the hypothesis that tenofovir treatment of osteoblasts causes changes in the gene expression profile that would impact osteoblast function during bone formation. Primary osteoblasts were isolated and then treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF). Total RNA from TDF-treated and untreated osteoblasts were extracted and used for microarray analysis to assess TDF-associated changes in the gene expression profile. Strikingly, the changes in gene expression profiles involved in cell signaling, cell cycle and amino acid metabolism, which would likely impact osteoblast function in bone formation. Our findings demonstrate for the first time that tenofovir treatment of primary osteoblasts results in gene expression changes that implicate loss of osteoblast function in tenofovir-associated bone mineral density loss.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/efeitos adversos , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Organofosfonatos/efeitos adversos , Osteoblastos/efeitos dos fármacos , Adenina/efeitos adversos , Adenina/farmacologia , Animais , Fármacos Anti-HIV/farmacologia , Densidade Óssea/genética , Reabsorção Óssea/genética , Camundongos , Camundongos Endogâmicos C57BL , Organofosfonatos/farmacologia , Osteoblastos/metabolismo , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacologia , TenofovirRESUMO
Clinical observations have implicated the antiretroviral drug tenofovir with bone density loss during the management of HIV infection. The goal of this study was to investigate the in vitro effects of tenofovir exposure of primary osteoclasts in order to gain insights into the potential mechanisms for the drug-induced bone density loss. We hypothesized that tenofovir may alter the expression of key genes involved in osteoclast function. To test this, primary osteoclasts were exposed to physiologically relevant concentrations of the prodrug tenofovir disoproxil fumarate (TDF), then intensive microarray analysis was done to compare tenofovir-treated versus untreated cells. Specific downregulation of Gnas, Got2 and Snord32a were observed in the TDF-treated cells. The functions of these genes help to explain the basis for tenofovir-associated bone density loss. Our studies represent the first analysis of the effects of tenofovir on osteoclast gene expression and help to explain the basis of tenofovir-associated bone density loss in HIV-infected individuals.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Aspartato Aminotransferase Mitocondrial/metabolismo , Densidade Óssea/efeitos dos fármacos , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Organofosfonatos/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoporose/induzido quimicamente , RNA Nucleolar Pequeno/metabolismo , Adenina/farmacologia , Animais , Aspartato Aminotransferase Mitocondrial/genética , Densidade Óssea/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromograninas , Regulação para Baixo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Expressão Gênica/efeitos dos fármacos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoclastos/metabolismo , Osteoporose/metabolismo , RNA Nucleolar Pequeno/genética , TenofovirRESUMO
Eukaryotic genomes contain either one or two genes encoding homologs of the highly conserved histone chaperone Asf1, however, little is known of their in vivo roles in animal development. UNC-85 is one of the two Caenorhabditis elegans Asf1 homologs and functions in post-embryonic replication in neuroblasts. Although UNC-85 is broadly expressed in replicating cells, the specificity of the mutant phenotype suggested possible redundancy with the second C. elegans Asf1 homolog, ASFL-1. The asfl-1 mRNA is expressed in the meiotic region of the germline, and mutants in either Asf1 genes have reduced brood sizes and low penetrance defects in gametogenesis. The asfl-1, unc-85 double mutants are sterile, displaying defects in oogenesis and spermatogenesis, and analysis of DNA synthesis revealed that DNA replication in the germline is blocked. Analysis of somatic phenotypes previously observed in unc-85 mutants revealed that they are neither observed in asfl-1 mutants, nor enhanced in the double mutants, with the exception of enhanced male tail abnormalities in the double mutants. These results suggest that the two Asf1 homologs have partially overlapping functions in the germline, while UNC-85 is primarily responsible for several Asf1 functions in somatic cells, and is more generally involved in replication throughout development.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Genes de Helmintos , Proteínas de Helminto/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Replicação do DNA , Transtornos do Desenvolvimento Sexual/genética , Embrião não Mamífero , Feminino , Proteínas de Helminto/genética , Histonas/genética , Hibridização In Situ , Masculino , Meiose , Modelos Biológicos , Chaperonas Moleculares/genética , Oogênese/genética , RNA Mensageiro/metabolismo , Espermatogênese/genéticaRESUMO
Normal animal development requires accurate cell divisions, not only in the early stages of rapid embryonic cleavages, but also in later developmental stages. The Caenorhabditis elegans unc-85 gene is implicated only in cell divisions that occur post-embryonically, primarily in terminal neuronal lineages. Variable post-embryonic cell division failures in ventral cord motoneuron precursors result in uncoordinated locomotion of unc-85 mutant larvae by the second larval stage. These neuroblast cell division failures often result in unequally sized daughter nuclei, and sometimes in nuclear fusions. Using a combination of conventional mapping techniques and microarray analysis, we cloned the unc-85 gene, and find that it encodes one of two C. elegans homologs of the yeast Anti-silencing function 1 (Asf1) histone chaperone. The unc-85 gene is expressed in replicating cells throughout development, and the protein is localized in nuclei. Examination of null mutants confirms that embryonic neuroblast cell divisions occur normally, but post-embryonic neuroblast cell divisions fail. Analysis of the DNA content of the mutant neurons indicates that defective replication in post-embryonic neuroblasts gives rise to ventral cord neurons with an average DNA content of approximately 2.5 n. We conclude that UNC-85 functions in post-embryonic DNA replication in ventral cord motor neuron precursors.