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PubMed2004

Effects of mutations in the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B on intracellular transport and infectivity.

Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.

Amino Acid Motifs↗
PubMed2000

Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation.

Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.

Base Sequence↗
PubMed1996

A Sendai virus vector leading to the efficient expression of mutant M proteins interfering with virus particle budding.

A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.

Animals↗
PubMed2003

Nuclear perturbations following adenovirus infection.

Adenoviruses are processed and assembled in the nuclei of infected cells and thereby produce significant perturbations to their structure and function. As the complex interactions that occur in the nuclei of uninfected cells are not yet fully understood many of the changes seen on infection have been described mainly in morphological terms. This chapter attempts to place more recent findings into this context and demonstrates that adenoviruses are able to hijack many cellular processes and enzymes to their advantage. In particular, modifications to nuclear PODs and nucleoli have more recently been explored in greater detail.

Adenoviridae Infections↗
PubMedDate not supplied

[The effect of fragment D on the aggregation of fibrinogen molecules in solution].

The mechanism of fibrinogen aggregation in the presence of fragment D has been studied. The values of translational diffusion coefficients, specific viscosity, average molecular masses, and Zimm factor of light scattering indicate that fragment D accelerated assembly of fibrinogen molecules that form flexible polymeric chains with tail-to-tail association due to spontaneous structural modification of COOH-terminal regions. Electrophoresis did not reveal the presence of fragment D in polymer fraction of non-reduced samples. The data obtained allowed to conclude that at initial stages fragment D forms unstable complexes with structurally modified fibrinogen molecules. These complexes serve as intermediates in the multistep process of assembly of supermolecular protein complex.

Blood Protein Electrophoresis↗
PubMed1981

A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein.

Morphogenesis of filamentous phage includes synthesis of the phage major coat protein in precursor form, its insertion into the host cell plasma membrane, its cleavage to the mature form of the protein, and its assembly there into virions. The M13 mutant am8H1R6 encodes a coat protein in which leucine replaces glutamic acid as residue 2 of the mature protein [Boeke, J. D., Russel, M. & Model, P. (1980) J. Mol. Biol. 144, 103-116]. The coat protein precursor produced by this variant is a poor substrate for the Escherichia coli signal peptidase both in vivo and in vitro. This pre-coat protein, which is eventually processed and assembled into viable phage particles, is associated with the membrane fraction of the infected cell. We conclude that the domain recognized by the signal peptidase extends beyond the signal peptide itself. Furthermore, membrane association and signal peptide cleavage can be separated temporally under conditions that permit membrane insertion, cleavage, and phage assembly.

Coliphages↗
PubMed1989

Amino acid sequence and length requirements for assembly and function of the colicin A lysis protein.

The roles of the various parts of the mature colicin A lysis protein (Cal) in its assembly into the envelope and its function in causing "quasi-lysis," the release of colicin A, and the activation of phospholipase A were investigated. By using cassette mutagenesis, many missense mutations were introduced into the highly conserved portion of the lysis protein. In vitro mutagenesis was also used to introduce stop codons after amino acids 16 and 18 and a frameshift mutation at amino acid 17 of the mature Cal sequence. The processing and modification of the mutants were identical to those of the wild type, except for the truncated Cal proteins, which were neither acylated nor processed. Thus, the carboxy-terminal half of Cal must be present (or replaced by another peptide) for the proper processing and assembly of the protein. However, the specific sequence of this region is not required for the membrane-damaging function of the protein. Furthermore, the sequence specificity for even the conserved amino acids of the amino-terminal half of the protein is apparently exceedingly relaxed, since only those mutant Cal proteins in which a highly conserved amino acid has been replaced by a glutamate were impaired in their function.

Amino Acid Sequence↗
PubMed2006

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids.

The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.

Actins↗
PubMed2003

The interplay of nuclear mRNP assembly, mRNA surveillance and export.

Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. Export of mRNAs is mediated by a conserved heterodimeric transport receptor (known as NXF1-p15 in metazoa) that binds to mRNA cargoes either directly or indirectly by means of adaptor proteins. Upon binding, the receptor translocates the cargo across the central channel of the nuclear pore complex (NPC) by interacting directly with NPC proteins called nucleoporins. Our understanding of the molecular mechanisms by which the heterodimeric receptor is recruited to cellular mRNAs indicates that the adaptors, together with additional proteins, are loaded cotranscriptionally to nascent mRNAs to form large ribonucleoprotein complexes (mRNPs). This mRNP assembly process might be subject to quality control by a nuclear mRNP surveillance mechanism, so that aberrantly assembled mRNPs are degraded before the receptor delivers them to the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed1991

Bovine papillomavirus type 1 alters the processing of host glucose- and calcium-modulated endoplasmic reticulum proteins.

We have previously characterized five proteins induced by the presence of the E2 open reading frame (ORF) region of bovine papillomavirus type 1 (BPV-1) in C127 mouse fibroblasts (R. M. Levenson, U. G. Brinckmann, M. K. O'Banion, E. J. Androphy, J. T. Schiller, F. Tabatabai, L. P. Turek, K. Neary, M. T. Chin, T. R. Broker, L. T. Chow, and D. A. Young, Virology 172:170-179, 1989). By specific immunoprecipitation, we now find that one of the papillomavirus-associated proteins (pvp1) is a highly glycosylated form of glucose-regulated protein 100 (grp100), a major constituent of the endoplasmic reticulum. A second set of pvps (2, 3, and 4) are shown to be related precursors of another protein already present in C127 cells (protein B). Based on their induction by the calcium ionophore A23187 and their positions on giant two-dimensional gels, we have tentatively identified pvp2, -3, and -4 and B as forms of calcium-regulated protein 55, another constituent of the endoplasmic reticulum (D. R. J. Macer and G. L. E. Koch, J. Cell Sci. 91:61-70, 1988). The mechanism by which BPV-1 brings about these changes is not yet defined; however, it is unlikely to involve calcium level perturbations or transformation per se, since ionophore treatment changes other proteins in C127 cells not seen with BPV and the papillomavirus-associated proteins are found in nontransformed cells harboring the E2 ORF region. Furthermore, the BPV changes are not associated with increased grp mRNA levels, as occurs in ionophore-treated cells. Rather, it appears that BPV-1 somehow retards the normal processing of these resident endoplasmic reticulum proteins that are believed to serve as critical regulators of host protein processing and assembly.

Animals↗
PubMed1989

ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.

Animals↗
PubMed2001

The delta and delta ' subunits of the DNA polymerase III holoenzyme are essential for initiation complex formation and processive elongation.

delta and delta' are required for assembly of the processivity factor beta(2) onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction. We developed protocols for generating highly purified preparations of delta and delta'. In holoenzyme reconstitution assays, delta' could not be replaced by delta, tau, or gamma, even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta could not be replaced by delta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of either the holA(delta) or holB(delta') genes were not viable, demonstrating that both delta and delta' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta and delta'. However, in the absence of chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta-delta' but not chipsi, delta, or delta' alone restored the diminished activity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, deltadelta' is present in initiation complex and is required for processive elongation.

Adenosine Triphosphate↗
PubMed1997

Structure of 20S proteasome from yeast at 2.4 A resolution.

The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

Acetylcysteine↗
PubMed1979

Regulation of the microtubule steady state in vitro by ATP.

ATP increases microtubule steady state assembly and disassembly rates in vitro in a concentration-dependent manner. Bovine brain microtubules, composed of 75% tubulin and 25% high molecular weight microtubule-associated proteins (MAPs), were purified by three cycles of assembly and disassembly in the absence of ATP. When assembled to steady state, these microtubules add dimers at one end and lose them at the other in a unidirectional assembly-disassembly process. In the presence of 1.0 mM ATP the unidirectional flow of tubulin from one end of the microtubules to the other increases as much as 20 fold, as revealed by loss of 3H-GTP from uniformly labeled microtubules under GTP chase conditions and by the rate of disassembly following addition of 50 microM podophyllotoxin. UTP, CTP and 5' adenylylimidodiphosphate (AMP-PNP) cannot substitute for ATP in producing this effect. Furthermore, the increase in steady state flow rate persists afer ATP is removed. Thus microtubules assembled in ATP and centrifuged through sucrose cushions to separate them from nucleotides continue to exhibit increased rates in the next assembly cycle in the absence of ATP. It is possible that an ATP-dependent microtubule protein kinase is responsible for the observed increase in tubulin flow rate. A kinase activity associated with brain MAPs has been reported to be cAMP-dependent (Sloboda et al., 1975). We have found an adenylate cyclase activity associated with these microtubules. Whether the adenylate cyclase is a contaminant or due to a specific microtubules-associated protein, and whether its activity is functionally linked to the increased rate of assembly and disassembly in the presence of ATP, remain to be determined.

Adenosine Triphosphate↗
PubMed2002

Using hierarchical self-assembly to form three-dimensional lattices of spheres.

This paper describes an approach to the fabrication of three-dimensional (3-D) structures of millimeter-scale spherical beads having a range of lattices-tetragonal, cubic, and hexagonal-using hierarchical self-assembly. The process has five steps: (i) metal-coated beads are packed in a rod-shaped cavity in an elastomeric polymer (poly(dimethylsiloxane), PDMS); (ii) the beads are embedded in a second polymer (PDMS or polyurethane, PU) using a procedure that leaves the parts of the beads in contact with the PDMS exposed; (iii) the exposed areas of the beads are coated with a solder having a low melting point; (iv) the polymer rods-with embedded beads and exposed solder drops-are suspended in an approximately isodense medium (an aqueous solution of KBr) and allowed to self-assemble by capillary interactions between the drops of molten solder; and (v) the assembly is finished by several procedures, including removing the beads from the polymer matrix by dissolution, filling the voids left with another material, and dissolving the matrix. The confinement of the beads in regular structures in polymer rods makes it possible to generate self-assembled structures with a variety of 3-D lattices; the type of the lattice formed can be controlled by varying the size of the beads, and the size and shape of the cross-section of the rods.

Journal Article↗
PubMed2003

Minimal cis-acting elements required for adenovirus genome packaging.

The design of drugs for treatment of virus infections and the exploitation of viruses as drugs for treatment of diseases could be made more successful by understanding the molecular mechanisms of virus-specific events. The process of assembly, and more specifically packaging of the genome into a capsid, is an obligatory step leading to future infections. To enhance our understanding of the molecular mechanism of packaging, it is necessary to characterize the viral components necessary for the event. In the case of adenovirus, sequences between nucleotides 200 and 400 at the left end of the genome are essential for packaging. This region contains a series of redundant bipartite sequences, termed A repeats, that function in packaging. Synthetic packaging sequences made of multimers of a single A repeat substitute for the authentic adenovirus packaging domain. A repeats are binding sites for the CCAAT displacement protein and the viral protein IVa2. Several lines of evidence implicate these proteins in the packaging process. It was not known, however, whether other cis-acting elements play a role in the packaging process as well. We utilized an in vivo approach to address the role of the inverted terminal repeats and the covalently linked terminal proteins in packaging of the adenovirus genome. Our results show that these elements are not necessary for efficient packaging of the viral genome. A significant implication of these results applicable to gene therapy vector design is that the linkage of the adenovirus packaging domain to heterologous DNA sequences should suffice for targeting to the viral capsid.

Adenoviruses, Human↗
PubMed1991

Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin.

Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [(35)S]cysteine, in which [(35)S]sulfide and iron are incorporated into Fd to build up the (35)S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [(35)S]sulfide or iron, or in Fe-S cluster formation itself. [(35)S]Sulfide was liberated from [(35)S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [(35)S]cysteine occurred before the formation of the (35)S-labeled Fe-S cluster, and the amount of radioactivity in [(35)S]sulfide was greater than that in (35)S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na(2)S) inhibited competitively formation of the (35)S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [(35)S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.

Journal Article↗
PubMed1976

Cycling aggregation patterns of cytoplasmic F-actin coordinated with oscillating tension force generation.

Isometric contracting protoplasmic veins of Physarum polycephalum show cycling patterns of cytoplasmic F-actin, dependent on their oscillating contraction behaviour (minute rhythms). The process of fibrillogenesis represents a parallel arrangement of F-actin chains ("plasma filaments, microfilaments") during the isometric contraction phase. A part of the results of the present work corroborates previous results on stretch-activated veins which showed that the fibrillar form of F-actin reflects the isometric contracted state. During isometric relaxation phase, a disaggregation of the fibrillar pattern takes place and is accompanied by a deparallelisation of F-actin chains. Therefore, the isometric relaxed state of cytoplasmic actomyosin is non-fibrillar in nature. Thus, the morphologically detectable fibrillar form of cytoplasmic actomyosin, according to physiological interpretation, is solely representative of the isometric contracted state. The question whether assembly-disassembly processes, e.g. G equilibrium F-actin-transformation, play a role in the contraction-relaxation cycle is discussed.

Actins↗