RESUMO
Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Dineínas do Citoplasma/genética , Dineínas/metabolismo , Vírus da Leucemia Murina/crescimento & desenvolvimento , Replicação Viral/genética , Células 3T3 , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular , Núcleo Celular/virologia , Dineínas/genética , Produtos do Gene gag/genética , Células HEK293 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos , Microtúbulos/metabolismoRESUMO
How murine leukemia virus (MLV) travels from the cell membrane to the nucleus and the mechanism for nuclear entry of MLV DNA in dividing cells still remain unclear. It seems likely that the MLV preintegration complex (PIC) interacts with cellular proteins to perform these tasks. We recently published that the microtubule motor cytoplasmic dynein complex and its regulator proteins interact with the MLV PIC at early times of infection, suggesting a functional interaction between the incoming viral particles, the dynein complex, and dynein regulators. To better understand the role of the dynein complex in MLV infection, we performed short hairpin RNA (shRNA) screening of the dynein light chains on MLV infection. We found that silencing of a specific light chain of the cytoplasmic dynein complex, DYNLRB2, reduced the efficiency of infection by MLV reporter viruses without affecting HIV-1 infection. Furthermore, the overexpression of DYNLRB2 increased infection by MLV. We conclude that the DYNLRB2 light chain of the cytoplasmic dynein complex is an important and specific piece of the host machinery needed for MLV infection.IMPORTANCE Retroviruses must reach the chromatin of their host to integrate their viral DNA, but first they must get into the nucleus. The cytoplasm is a crowded environment in which simple diffusion is slow, and thus viruses utilize retrograde transport along the microtubule network, mediated by the dynein complex. Different viruses use different components of this multisubunit complex. We have found that murine leukemia virus (MLV) associates functionally and specifically with the dynein light chain DYNLRB2, which is required for infection. Our study provides more insight into the molecular requirements for retrograde transport of the MLV preintegration complex and demonstrates, for the first time, a role for DYNLRB2 in viral infection.
Assuntos
Dineínas do Citoplasma/genética , Dineínas do Citoplasma/fisiologia , Interações Hospedeiro-Patógeno , Vírus da Leucemia Murina/fisiologia , Animais , Transporte Biológico , Linhagem Celular , Núcleo Celular/virologia , Células HEK293 , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Microtúbulos/virologia , Células NIH 3T3RESUMO
Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport. Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD). D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1. We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants. Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry. As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD. However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM. Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.
Assuntos
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Lipoproteínas/metabolismo , Substituição de Aminoácidos , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Linhagem Celular , Colesterol/química , Colesterol/genética , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
In this study, the effect of α-eleostearic acid (α-ESA) on the lipid peroxidation of soybean asolectin (ASO) liposomes was investigated. This effect was correlated to changes caused by the fatty acid in the membrane dynamics. The influence of α-ESA on the dynamic properties of liposomes, such as hydration, mobility and order, were followed by horizontal attenuated total reflection Fourier transform infrared spectroscopy (HATR-FTIR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and UV-vis techniques. The α-ESA showed an in vitro antioxidant activity against the damage induced by hydroxyl radical (OH) in ASO liposomes. The analysis of HATR-FTIR frequency shifts and bandwidths and (1)H NMR spin-lattice relaxation times, related to specific lipid groups, showed that α-ESA causes an ordering effect on the polar and interfacial regions of ASO liposomes, which may restrict the OH diffusion in the membrane. The DSC enthalpy variation analysis suggested that the fatty acid promoted a disordering effect on lipid hydrophobic regions, which may facilitate interactions between the reactive specie and α-ESA. Turbidity results showed that α-ESA induces a global disordering effect on ASO liposomes, which may be attributed to a change in the lipid geometry and shape. Results of this study may allow a more complete view of α-ESA antioxidant mode of action against OH, considering its influence on the membrane dynamics.
Assuntos
Antioxidantes/farmacologia , Glycine max/química , Ácidos Linolênicos/farmacologia , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Termodinâmica , Antioxidantes/química , Radical Hidroxila/antagonistas & inibidores , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Ácidos Linolênicos/química , Peroxidação de Lipídeos/efeitos dos fármacos , Lipossomos/química , Fosfatidilcolinas/química , Glycine max/metabolismoRESUMO
Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.
Assuntos
Venenos de Cnidários/farmacologia , Lipídeos/química , Microdomínios da Membrana/metabolismo , Anêmonas-do-Mar/metabolismo , Animais , Varredura Diferencial de Calorimetria , Microscopia de Força Atômica , Compostos Orgânicos/farmacologia , Anêmonas-do-Mar/químicaRESUMO
Caiman latirostris is a reptilian species that exhibits temperature-dependent sex determination (TSD). Male-to-female sex reversal can be achieved after in ovo estrogen/xenoestrogen exposure. This is known as hormone-dependent sex determination (HSD). The amh, sox9 and sf-1 genes are involved in sex determination, sex differentiation, and steroidogenesis. The aims of this study were: (a) to establish the expression patterns of amh, sox9 and sf-1 mRNA in the gonad-adrenal-mesonephros (GAM) complexes of neonatal TSD-male and TSD-female caimans, (b) to compare the expression of these genes between TSD-females and HSD-females (born from E2-exposed eggs incubated at the male-producing temperature) and (c) to evaluate whether in ovo exposure to a low dose of E2 or bisphenol A (BPA) or to a high dose of endosulfan (END) modifies amh, sox9 or sf-1 mRNA expressions in neonatal males. The mRNA expressions of amh, sox9 and sf-1 in GAM complexes from TSD-males and TSD-females and from HSD-females were quantitatively compared by RT-PCR. A sexually dimorphic pattern of amh and sox9 mRNA expression was found, with a higher expression in TSD-males than in TSD-females. sf-1 mRNA did not differ between TSD-males and TSD-females. HSD-females exhibited a higher expression of sox9 than TSD-females. In males, increased mRNA expression of sex-determining genes was observed after in ovo exposure to END. E2 decreased sox9 but increased sf-1 mRNA expression. Changes induced by BPA were evident although not significant. These results provide new insights into the potential mechanisms that lead to the gonadal histo-functional alterations observed in caimans exposed to contaminated environments.