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Evidence for a functional link between uncoating of the human immunodeficiency virus type 1 core and nuclear import of the viral preintegration complex.
Dismuke DJ, Aiken C
(2006) J Virol 80: 3712-20
MeSH Terms: Active Transport, Cell Nucleus, Cell Line, DNA, Viral, HIV Long Terminal Repeat, HIV-1, Humans, Transcription, Genetic, Viral Core Proteins, Viral Regulatory and Accessory Proteins, Virus Integration
Show Abstract · Added February 19, 2015
Human immunodeficiency virus type 1 (HIV-1) particles begin their replication upon fusion with the plasma membrane of target cells and release of the viral core into the host cell cytoplasm. Soon thereafter, the viral capsid, which is composed of a polymer of the CA protein, disassociates from the internal ribonucleoprotein complex. While this disassembly process remains poorly understood, the available evidence indicates that proper uncoating of the core is a key step in infection. Defects in uncoating most often lead to a failure of the virus to undergo reverse transcription, resulting in an inability to form a functional viral preintegration complex (PIC). In a previous study, we reported that an HIV-1 mutant containing two substitutions in CA (Q63A/67A) was unusual in that it was poorly infectious yet synthesized normal levels of viral DNA. Here we report that this mutant is impaired for nuclear entry. Quantitative analysis of viral DNA synthesis from infected cells by Southern blotting and real-time PCR revealed that the Q63A/Q67A mutant is impaired in the synthesis of one-long terminal repeat (1-LTR) and 2-LTR circles. Isolation of PICs from acutely infected cells revealed that the Q63A/Q67A mutant produces protein-DNA complexes similar to wild-type in yield and overall composition, but these PICs contained elevated levels of CA and were impaired for integration in vitro. These results demonstrate that mutations in CA can have deleterious effects on both nuclear targeting and integration, suggesting that these steps in the HIV-1 life cycle are dependent on proper uncoating of the viral core.
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10 MeSH Terms
Reovirus variants selected for resistance to ammonium chloride have mutations in viral outer-capsid protein sigma3.
Clark KM, Wetzel JD, Gu Y, Ebert DH, McAbee SA, Stoneman EK, Baer GS, Zhu Y, Wilson GJ, Prasad BV, Dermody TS
(2006) J Virol 80: 671-81
MeSH Terms: Adaptation, Physiological, Ammonium Chloride, Animals, Capsid Proteins, Cathepsin L, Cathepsins, Cysteine Endopeptidases, Cysteine Proteinase Inhibitors, Drug Resistance, Viral, L Cells, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Reoviridae, Serial Passage, Viral Proteins, Viral Regulatory and Accessory Proteins, Virus Assembly
Show Abstract · Added December 10, 2013
Mammalian reoviruses are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis resulting in removal of the sigma3 protein and proteolytic cleavage of the mu1/mu1C protein. Ammonium chloride (AC) is a weak base that blocks disassembly of reovirus virions by inhibiting acidification of intracellular vacuoles. To identify domains in reovirus proteins that influence pH-sensitive steps in viral disassembly, we adapted strain type 3 Dearing (T3D) to growth in murine L929 cells treated with AC. In comparison to wild-type (wt) T3D, AC-adapted (ACA-D) variant viruses exhibited increased yields in AC-treated cells. AC resistance of reassortant viruses generated from a cross of wt type 1 Lang and ACA-D variant ACA-D1 segregated with the sigma3-encoding S4 gene. The deduced sigma3 amino acid sequences of six independently derived ACA-D variants contain one or two mutations each, affecting a total of six residues. Four of these mutations, I180T, A246G, I347S, and Y354H, cluster in the virion-distal lobe of sigma3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant sigma3 proteins. In comparison to wt virions, ACA-D viruses displayed enhanced susceptibility to proteolysis by endocytic protease cathepsin L. Image reconstructions of cryoelectron micrographs of three ACA-D viruses that each contain a single mutation in the virion-distal lobe of sigma3 demonstrated native capsid protein organization and minimal alterations in sigma3 structure. These results suggest that mutations in sigma3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.
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19 MeSH Terms
Reovirus sigma NS and mu NS proteins form cytoplasmic inclusion structures in the absence of viral infection.
Becker MM, Peters TR, Dermody TS
(2003) J Virol 77: 5948-63
MeSH Terms: Animals, Cell Line, HeLa Cells, Humans, Inclusion Bodies, Viral, L Cells, Mice, Precipitin Tests, Reoviridae, Subcellular Fractions, Transfection, Two-Hybrid System Techniques, Viral Nonstructural Proteins, Viral Proteins, Viral Regulatory and Accessory Proteins, Virus Replication
Show Abstract · Added December 10, 2013
Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, sigma NS and micro NS, and structural protein sigma 3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed sigma NS, mu NS, and sigma 3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, sigma NS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed sigma NS is functional as it complements the defect in temperature-sensitive, sigma NS-mutant virus tsE320. In both transfected and infected cells, mu NS is found in punctate cytoplasmic structures and sigma 3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of mu NS and sigma 3 is not altered when the proteins are expressed together or with sigma NS. However, when expressed with micro NS, sigma NS colocalizes with mu NS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both sigma NS and mu NS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, sigma NS interacts with mu NS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that sigma NS and mu NS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.
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16 MeSH Terms
Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1.
Lopez CF, Montal M, Blasie JK, Klein ML, Moore PB
(2002) Biophys J 83: 1259-67
MeSH Terms: Carbon, Cell Membrane, Computer Simulation, Electrons, HIV-1, Human Immunodeficiency Virus Proteins, Lipid Bilayers, Magnetic Resonance Spectroscopy, Models, Molecular, Octanes, Phosphatidylethanolamines, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Software, Time Factors, Viral Regulatory and Accessory Proteins, Water
Show Abstract · Added March 10, 2014
Molecular dynamics (MD) simulations have been carried out on bundles of the channel-forming transmembrane (TM) domain of the viral protein U (VPU(1-27) and VPU(6-27)) from the human immunodeficiency virus (HIV-1). Simulations of hexameric and pentameric bundles of VPU(6-27) in an octane/water membrane mimetic system suggested that the pentamer is the preferred oligomer. Accordingly, an unconstrained pentameric helix bundle of VPU(1-27) was then placed in a hydrated palmitoyl-oleyl-3-n-glycero-phosphatidylethanolamine (POPE) lipid bilayer and its structural properties calculated from a 3-ns MD run. Some water molecules, initially inside the channel lumen, were expelled halfway through the simulation and the bundle adopted a conical structure reminiscent of previous MD results obtained for VPU(6-27) in an octane/water system. The pore constriction generated may correspond to a closed state of the channel and underlies the relocation of the W residue toward the pore lumen. The relative positions of the helices with respect to the bilayer and their interactions with the lipids are discussed. The observed structure is stabilized via specific interactions between the VPU helices and the carbonyl oxygen atoms of the lipid molecules, particularly at the Q and S residues.
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18 MeSH Terms
Sequence diversity within the reovirus S3 gene: reoviruses evolve independently of host species, geographic locale, and date of isolation.
Goral MI, Mochow-Grundy M, Dermody TS
(1996) Virology 216: 265-71
MeSH Terms: Animals, Base Sequence, Biological Evolution, Cell Line, Genetic Variation, Geography, Humans, L Cells, Mice, Molecular Sequence Data, Phylogeny, Reoviridae, Sequence Homology, Amino Acid, Serotyping, Viral Proteins, Viral Regulatory and Accessory Proteins
Show Abstract · Added December 10, 2013
To better understand genetic diversity of mammalian reoviruses, we studied sequence variability in the S3 gene segment of 17 field-isolate reovirus strains and prototype strains of the three reovirus serotypes. Strains studied were isolated over a 37-year period from different mammalian hosts and geographic locations. A high degree of variability was observed in the nucleotide sequences of the S3 gene, whereas the deduced amino acid sequences of the S3 gene product, sigma NS, were highly conserved. When variability among the S3 nucleotide sequences was analyzed using pairwise comparisons, we found that 5' and 3' noncoding regions were significantly more conserved than the remainder of the gene. This high degree of sequence conservation was also observed within the first 15 nucleotides of the 5' coding region. Phylogenetic analyses showed that multiple alleles of the S3 gene cocirculate and that genetic diversity in the S3 gene does not correlate with host species, geographic locale, or date of isolation. Phylogenetic trees constructed from variation in the S3 sequences are distinct from those previously generated from sequences that encode attachment protein sigma 1, core protein sigma 2, and outer capsid protein sigma 3, which supports the hypothesis that reovirus gene segments reassort in nature. These findings suggest that reovirus gene segments are well-adapted to mammalian hosts and that reovirus evolution has reached an equilibrium.
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16 MeSH Terms
Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambda repressor.
Breyer RM, Sauer RT
(1989) J Biol Chem 264: 13355-60
MeSH Terms: Amino Acid Sequence, Antibodies, Monoclonal, Antibodies, Viral, Antibody Specificity, Antigen-Antibody Complex, Bacteriophage lambda, Base Sequence, Binding Sites, Antibody, DNA Mutational Analysis, DNA-Binding Proteins, Molecular Sequence Data, Protein Conformation, Repressor Proteins, Transcription Factors, Viral Proteins, Viral Regulatory and Accessory Proteins
Show Abstract · Added December 21, 2013
Monoclonal antibody 51F recognizes determinants in the helix 4 region of the native form of the N-terminal domain of lambda repressor. A cassette mutagenesis method was used to introduce changes within this region, and antibody-reactive candidates were isolated and sequenced. The resulting data allow the identification of repressor side chains that are critical determinants of antibody binding. Four of these side chains are on the surface of the N-terminal domain and probably contact the antibody directly. These contact positions were then mutagenized individually, and the antibody binding phenotypes of a large number of singly mutant repressors were determined. Taken together, the mutational data allow a functional map of the recognition surface to be constructed and the physical nature of some of the specific interactions that stabilize the antibody-antigen complex to be surmised.
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16 MeSH Terms
Production and characterization of monoclonal antibodies to the N-terminal domain of lambda repressor.
Breyer RM, Sauer RT
(1989) J Biol Chem 264: 13348-54
MeSH Terms: Amino Acid Sequence, Animals, Antibodies, Monoclonal, Antibodies, Viral, Bacteriophage lambda, Binding Sites, Antibody, Binding, Competitive, DNA-Binding Proteins, Female, Mice, Mice, Inbred BALB C, Protein Conformation, Repressor Proteins, Transcription Factors, Viral Proteins, Viral Regulatory and Accessory Proteins
Show Abstract · Added December 21, 2013
Monoclonal antibodies reactive with distinct regions of the N-terminal domain of the lambda repressor protein have been isolated. By comparing the affinities of these antibodies for mutant repressors with increased and decreased thermal stabilities, each of the antibodies can be shown to bind to epitopes accessible in the native conformation of the N-terminal domain. Experiments probing antibody binding to protein fragments, mutant variants, and peptides have also been used to define likely regions of contact between the antibodies and the N-terminal domain.
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16 MeSH Terms