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Human immunodeficiency virus type-1 reverse transcriptase and ribonuclease h as substrates of the viral protease
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Alfredo G. Tomasselli Jean L. Sarcich Linda J. Barrett Ilene M. Reardon W. Jeffrey Howe David B. Evans Satish K. Sharma Robert L. Heinrikson 《Protein science : a publication of the Protein Society》1993,2(12):2167-2176
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Low pH is required for avian sarcoma and leukosis virus Env-dependent viral penetration into the cytosol and not for viral uncoating
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A novel entry mechanism has been proposed for the avian sarcoma and leukosis virus (ASLV), whereby interaction with specific cell surface receptors activates or primes the viral envelope glycoprotein (Env), rendering it sensitive to subsequent low-pH-dependent fusion triggering in acidic intracellular organelles. However, ASLV fusion seems to proceed to a lipid mixing stage at neutral pH, leading to the suggestion that low pH might instead be required for a later stage of viral entry such as uncoating (L. J. Earp, S. E. Delos, R. C. Netter, P. Bates, and J. M. White. J. Virol. 77:3058-3066, 2003). To address this possibility, hybrid virus particles were generated with the core of human immunodeficiency virus type 1 (HIV-1), a known pH-independent virus, and with subgroups A or B ASLV Env proteins. Infection of cells by these pseudotyped virions was blocked by lysosomotropic agents, as judged by inhibition of HIV-1 DNA synthesis. Furthermore, by using HIV-1 cores that contain a Vpr-beta-lactamase fusion protein (Vpr-BlaM) to monitor viral penetration into the cytosol, we demonstrated that virions bearing ASLV Env, but not HIV-1 Env, enter the cytosol in a low-pH-dependent manner. This effect was independent of the presence of the cytoplasmic tail of ASLV Env. These studies provide strong support for the model, indicating that low pH is required for ASLV Env-dependent viral penetration into the cytosol and not for viral uncoating. 相似文献
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Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8-9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin. 相似文献
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Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase. 总被引:9,自引:0,他引:9
One- and two-diminsional tryptic and chymotryptic peptide maps of 125-I-labeled alpha and alphabeta avian myeloblastosis virus DNA polymerase demonstrate that the alpha polypeptide of the one and two subunit enzymes are structurally similar, if not identical. Furthermore, the beta subunit contains the same major 125I-labeled peptides as alpha, plus several additional peptides. These relationships and the fact that aging of purified alphabeta avian myeloblastosis virus DNA polymerase increases the proportion of alpha DNA polymerase that can be isolated from the alphabeta enzyme by phosphocellulose chromatography, suggests that alpha is derived from beta by proteolytic cleavage. 相似文献
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Influence of phosphate on activity and stability of reverse transcriptase from avian myeloblastosis virus. 总被引:4,自引:0,他引:4
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Activity of RNA-dependent DNA polymerase (RDDP) from avian myeloblastosis virus (AMV), either in purified form or in virus lysates, was increased by phosphorylation. Stability of RDDP in lysates buffered with phosphate was much greater (no loss of activity in 48 hours at 4 degrees) than that in lysates buffered with Tris-Cl (76% loss). Activity lost in the Tris-buffered extracts was completely restored by phosphorylation. The findings suggested that AMV RDDP activity is influenced by the degree of phosphorylation of the enzyme or enzyme-associated proteins and that this chemical modification is mediated by protein phosphokinase and phosphoprotein phosphatase present in crude extracts of purified AMV. Application of these results provided the basis of procedures whereby RDDP can be recovered in significantly higher yield and purity than formerly. 相似文献
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Dirk Jochmans Maria Anders Inge Keuleers Liesbeth Smeulders Hans-Georg Kräusslich Günter Kraus Barbara Müller 《Retrovirology》2010,7(1):1-14
Background
The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.Results
In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.Conclusions
It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD. 相似文献17.
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