Biomedical subjects

E Barklis

Publications and source records attributed to E Barklis.

At least 19 recordsLinked to original sources

PubMed2001

Hantavirus nucleocapsid protein oligomerization.

Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420- to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates.

Amino Acid Motifs↗

PubMed2000

Three-dimensional organization of retroviral capsid proteins on a lipid monolayer.

We have used a method for the two-dimensional crystallization of retroviral structural proteins to obtain a three-dimensional structure of negatively stained, membrane-bound, histidine-tagged Moloney murine leukemia virus (M-MuLV) capsid protein (his-MoCA) arrays. Tilted and untilted micrographs from crystals formed by purified his-MoCA proteins incubated beneath lipid monolayers containing nickel-chelating lipids were used in 3D reconstructions. The 2D crystals had unit cell dimensions of a=72.6 A, b=72.5 A and gamma=119.5 degrees, but appeared to have no intrinsic symmetry (p1) in 3D, in contrast to the trigonal or hexagonal appearance of their 2D projections. Membrane-bound his-MoCA proteins showed a strand-like organization, apparently with dimer building blocks. Membrane-proximal regions, or putative N-terminal domains (NTDs), dimerized with different partners than the membrane-distal putative C-terminal domains (CTDs). Evidence also suggests that CTDs can adopt alternate orientations relative to their NTDs, forming interstrand connections. Our results are consistent with helical-spiral models for retrovirus particle assembly, but are not easily reconcilable with icosahedral models.

Capsid↗

PubMed2000

Crosslink analysis of N-terminal, C-terminal, and N/B determining regions of the Moloney murine leukemia virus capsid protein.

To analyze contacts made by Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins in immature and mature virus particles, we have employed a cysteine-specific crosslinking approach that permits the identification of retroviral Gag protein interactions at particular residues. For analysis, single cysteine creation mutations were made in the context of protease-deficient or protease-competent parental constructs. Cysteine creation mutations were chosen near the N- and C-termini of CA and at a site adjacent to the M-MuLV Glu-Ala Fv1 N/B host range determination sequence. Analysis of immature virions showed that PrGag proteins were crosslinked at C-terminal CA residues to form dimers while crosslinking of particle-associated N-terminal and N/B region mutant proteins did not yield dimers, but showed evidence of linking to an unknown 140- to 160-kDa partner. Analysis of mature virions demonstrated that both N- and C-terminal CA residues participated in dimer formation, suggesting that processed CA N- and C-termini are free to establish interprotein associations. Interestingly, N/B region mutant residues in mature virus particles did not crosslink to form dimers, but showed a novel crosslinked band, consistent with an interaction between the N/B tropism determining region and a cellular protein of 45-55 kDa.

Animals↗

PubMed2000

Atomic force microscopy and electron microscopy analysis of retrovirus Gag proteins assembled in vitro on lipid bilayers.

We have used an in vitro system that mimics the assembly of immature Moloney murine leukemia virus (M-MuLV) particles to examine how viral structural (Gag) proteins oligomerize at membrane interfaces. Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) were obtained on membrane bilayers composed of phosphatidylcholine (PC) and the nickel-chelating lipid 1, 2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'N-(5-amino-1-carboxy pentyl)iminodiacetic acid)propyl ether (DHGN). The membrane-bound arrays were analyzed by electron microscopy (EM) and atomic force microscopy (AFM). Two-dimensional projection images obtained by EM showed that bilayer-bound his-MoCA proteins formed cages surrounding different types of protein-free cage holes with similar cage holes spaced at 81.5-A distances and distances between dissimilar cage holes of 45.5 A. AFM images, showing topological features viewed near the membrane-proximal domain of the his-MoCA protein, revealed a cage network of only symmetrical hexamers spaced at 79-A distances. These results are consistent with a model in which dimers constitute structural building blocks and where membrane-proximal and distal his-MoCA regions interact with different partners in membrane-bound arrays.

Capsid↗

PubMed2000

Assembly of retrovirus capsid-nucleocapsid proteins in the presence of membranes or RNA.

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

Amino Acid Sequence↗

PubMed1998

Solution structures of human immunodeficiency virus type 1 (HIV-1) and moloney murine leukemia virus (MoMLV) capsid protein major-homology-region peptide analogs by NMR spectroscopy.

The capsid domain of retroviral Gag proteins possesses a single highly conserved subdomain termed the major homology region (MHR). While the mutagenesis of residues in the MHR will impair virus infectivity, the precise solution structure and function of the MHR is not known. To aid the structure/function characterization of the MHR, the structures of synthetic peptides encompassing the MHR of the human immunodeficiency virus type I (HIV-1) and Moloney murine leukemia virus (MoMLV) capsid proteins were investigated by several techniques. Homology-based secondary-structure prediction suggested that the HIV-1 and MoMLV peptides could form 50% and 38% alpha-helix, respectively. CD studies indicated that, in the presence of 50% trifluoroethanol, the HIV-1 peptide adopts an alpha-helical structure over half of its length, while the MoMLV peptide is over one third alpha-helix. Further analysis by 1H-NMR suggested that the C-terminal portion of the MHR of each virus forms a helix in aqueous solution. Distance-geometry structures of each peptide were calculated from NOE distance restraints and were refined by restrained molecular dynamics. The C-terminal halves of both peptides were observed to be in an alpha-helical conformation, while the N-terminal halves were disordered. Furthermore, both helices were amphipathic with high conservation of amino acid side-chain character, suggesting that a conserved helical MHR C-terminus is essential to retroviral capsid protein function.

Amino Acid Sequence↗

PubMed1998

Organization of HIV-1 capsid proteins on a lipid monolayer.

In an in vitro system that mimics the assembly of immature human immunodeficiency virus (HIV) particles, ordered arrays of HIV-1 capsid (CA) proteins encoded by the viral gag gene have been obtained by incubation of histidine-tagged capsid proteins (His-HIVCA) beneath lipid monolayers containing the nickel-chelating lipid, 1,2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'-N-(5-amino-1- carboxypentyl)iminodiacetic acid)propyl ether. The membrane-bound His-HIVCA proteins formed small crystalline arrays of primitive (p1) unit cells with dimensions of a = 74.2 A, b = 126.2 A, gamma = 89.3 degrees. The image-analyzed two-dimensional projection of His-HIVCA assemblies shows a cage-like lattice, consisting of hexamer and trimer units, surrounding protein-free cage holes. The hexamer-coordinated cage holes of 26.3-A diameter are spaced at 74. 2-A intervals: these distances, and the hexamer-trimer arrangement, are consistent with previous, lower resolution studies on immature HIV-1 virus particles produced in vivo. Additionally, HIV-1 matrix protein trimer unit structures align to the His-HIVCA trimer units such that residues previously shown to interact with the HIV-1 gp120/gp41 envelope protein complex are oriented toward the hexamer cage holes. Our results form a bridge between results from conventional methods for the analysis of HIV particle structure.

Capsid↗

PubMed1998

Analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain.

Previous studies have shown that in addition to its function in specific RNA encapsidation, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is required for efficient virus particle assembly. However, the mechanism by which NC facilitates the assembly process is not clearly established. Formally, NC could act by constraining the Pr559gag polyprotein into an assembly-competent conformation or by masking residues which block the assembly process. Alternatively, the capacity of NC to bind RNA or make interprotein contacts might affect particle assembly. To examine its role in the assembly process, we replaced the NC domain in Pr55gag with polypeptide domains of known function, and the chimeric proteins were analyzed for their abilities to direct the release of virus-like particles. Our results indicate that NC does not mask inhibitory domains and does not act passively, by simply providing a stable folded monomeric structure. However, replacement of NC by polypeptides which form interprotein contacts permitted efficient virus particle assembly and release, even when RNA was not detected in the particles. These results suggest that formation of interprotein contacts by NC is essential to the normal HIV-1 assembly process.

Animals↗

PubMed1997

Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C.

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GP) anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysaPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca(2+)-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein.

3T3 Cells↗

PubMed1997

Structural analysis of membrane-bound retrovirus capsid proteins.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.

Amino Acid Sequence↗

PubMed1997

A role for two hairpin structures as a core RNA encapsidation signal in murine leukemia virus virions.

Four putative hairpin structures (hairpins A to D) are involved in the specific encapsidation of Moloney murine leukemia virus (M-MuLV) RNA into M-MuLV virus particles. The C and D elements, encompassing M-MuLV viral nucleotides 310 to 374, facilitate encapsidation of heterologous RNA into virions. Thus, these two elements appear to act as a core RNA encapsidation signal. The loop sequences of the putative C and D hairpins are identical (GACG). However, when GACG loops were introduced into RNAs on heterologous stem sequences, they increased encapsidation levels only three- to fourfold. These results suggest that C and D stem-and-loop sequences contribute to the M-MuLV cis-acting site for encapsidation.

3T3 Cells↗

PubMed1997

Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation.

The human immunodeficiency virus (HIV) Pr55Gag precursor proteins direct virus particle assembly. While Gag-Gag protein interactions which affect HIV assembly occur in the capsid (CA) domain of Pr55Gag, the nucleocapsid (NC) domain, which functions in viral RNA encapsidation, also appears to participate in virus assembly. In order to dissect the roles of the NC domain and the p6 domain, the C-terminal Gag protein domain, we examined the effects of NC and p6 mutations on virus assembly and RNA encapsidation. In our experimental system, the p6 domain did not appear to affect virus release efficiency but p6 deletions and truncations reduced the specificity of genomic HIV-1 RNA encapsidation. Mutations in the nucleocapsid region reduced particle release, especially when the p2 interdomain peptide or the amino-terminal portion of the NC region was mutated, and NC mutations also reduced both the specificity and the efficiency of HIV-1 RNA encapsidation. These results implicated a linkage between RNA encapsidation and virus particle assembly or release. However, we found that the mutant ApoMTRB, in which the nucleocapsid and p6 domains of HIV-1 Pr55Gag were replaced with the Bacillus subtilis MtrB protein domain, released particles efficiently but packaged no detectable RNA. These results suggest that, for the purposes of virus-like particle assembly and release, NC can be replaced by a protein that does not appear to encapsidate RNA.

Animals↗

PubMed1996

Spectroscopic study of an HIV-1 capsid protein major homology region peptide analog.

The capsid (CA) domain of retroviral Gag proteins possesses one subdomain, the major homology region (MHR), which is conserved among nearly all avian and mammalian retroviruses. While it is known that the mutagenesis of residues in the MHR will impair virus infectivity, the precise structure and function of the MHR is not known. In order to obtain further information on the MHR, we have examined the structure of a synthetic peptide encompassing the MHR of human immunodeficiency virus type I (HIV-1) CA protein. Multiple sequence alignment and secondary structure prediction indicate that the peptide could form 50% alpha-helix and 10% beta-sheet. In addition, circular dichroism studies indicate that, in the presence of 50% trifluoroethanol (TFE), the peptide adopts an alpha-helical structure over half of its length. Further analysis by proton nuclear magnetic resonance spectroscopy suggests that the C-terminal portion of the MHR forms a helix in aqueous solution. Upon the addition of TFE, the position of the helix remains nearly constant, but the magnitude of the changes in H alpha chemical shifts of the residues indicate a more stable helix. These results suggest that a helical C-terminus of retroviral MHRs may be integral to the function of this region.

Amino Acid Sequence↗

PubMed1996

cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA.

We have analyzed the roles of RNA structural motifs located in the 5' part of the Moloney murine leukemia virus (M-MuLV) encapsidation domain (Psi region) with regard to their effects on viral replication. Four putative stem-loop structures between the 5' splice donor site and the gag initiation codon have been examined: stem structure A, corresponding to M-MuLV viral nucleotides 211 to 224; stem-loop B, nucleotides 278 to 303; stem-loop C, nucleotides 310 to 352; and stem-loop D, nucleotides 355 to 374. By measuring infectivities, encapsidation and splicing efficiencies, and endogenous reverse transcription levels of motif A, B, C, and D deletion mutants, we identified mutations which affect replication at the encapsidation step. In particular, deletion of all four motifs in a single mutant eliminated encapsidation of viral RNA, while deletion of individual elements moderately reduced the encapsidation efficiencies. Through analysis of different deletion combinations, we found that deletion of the first two motifs (A plus B) reduced both encapsidation and reverse transcription efficiencies, while deletion of the 3' motifs (C plus D) eliminated encapsidation. Interestingly, the C and D motifs both contain a GACG loop sequence and are highly conserved among murine type C retroviruses. Our results indicate that M-MuLV motifs C and D are necessary for efficient encapsidation, and the presence of at least one of these two stem-loops is crucial to encapsidation and virus replication.

Base Sequence↗

PubMed1996

Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents.

We have examined structural interactions of Gag proteins in human immunodeficiency virus type 1 (HIV-1) particles by utilizing cysteine mutagenesis and cysteine-specific modifying reagents. In immature protease-minus but otherwise wild-type (wt) particles, precursor Pr55Gag proteins did not form intermolecular cystines naturally but could be cross-linked at cysteines, and cross-linking appeared to occur across nucleocapsid (NC) domains. Capsid (CA) proteins in wt mature viruses possess cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bonds, nor can they be intermolecularly cross-linked by using the membrane-permeable cross-linker bis-maleimido hexane. The cysteine at gag codon 350 (C-350) is highly reactive to thiol-specific modifying reagents, while the one at codon 330 (C-330) appears considerably less reactive, even in the presence of ionic detergent. These results suggest that the HIV-1 CA C terminus forms an unusually stable conformation. Mutagenesis of C-350 to a serine residue in the mutant C350S (C-350 changed to serine) virtually eliminated particle assembly, attesting to the importance of this region. We also examined a C330S mutant, as well as mutants in which cysteines were created midway through the capsid domain or in the C-terminal section of the major homology region. All such mutants appeared wt on the basis of biochemical assays but showed greatly reduced infectivities, indicative of a postassembly, postprocessing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, implying either that these mutations permit cross-linking of the native C-terminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.

Animals↗

PubMed1995

Stem cell factor binding to retrovirus primer binding site silencers.

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and overlaps the Moloney murine leukemia virus (M-MuLV) proline primer binding site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silencer. Factor A bound specifically to the wild-type silencer element at room temperature and 30 degrees C, but not at 4 degrees C, and bound 10-fold better to the full-length silencer than to a minimal silencer core element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiated cells in which the silencer is nonfunctional. Salt and ion requirements for factor A binding were investigated, and partial purification steps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silencer and PBS activities, sequences representing phenylalanine, isoleucine, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell-specific repression of M-MuLV expression and in vitro in DNA binding assays. Of these PBS elements, only the lysine-1,2 PBS DNA fragment showed consistently high levels of repression. Interestingly, the lysine-1,2 PBS DNA fragment also formed a complex with an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding studies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna virus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited in undifferentiated stem cells.

Base Sequence↗

PubMed1995

Structural interactions between retroviral Gag proteins examined by cysteine cross-linking.

We have examined structural interactions between Gag proteins within Moloney murine leukemia virus (M-MuLV) particles by making use of the cysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65Gag proteins in immature particles were intermolecularly cross-linked at cysteines to form Pr65Gag oligomers, from dimers to pentamers or hexamers. Following a systematic approach of cysteine-to-serine mutagenesis, we have shown that cross-linking of Pr65Gag occurred at cysteines of the nucleocapsid (NC) Cys-His motif, suggesting that the Cys-His motifs within virus particles are packed in close proximity. The M-MuLV Pr65Gag protein did not cross-link to the human immunodeficiency virus Pr55Gag protein when the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close enough proximity to be cross-linked. Using an assembly-competent, protease-minus, cysteine-minus Pr65Gag protein as a template, novel cysteine residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showed above-background levels of Gag-Gag dimers but also identified a novel cellular factor, present in virions, that cross-linked to MHR residues. Although the NC cysteine mutation was compatible with M-MuLV particle assembly, deletions of the NC domain were not tolerated. These results suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or non-Cys-His motif domains of the NC.

3T3 Cells↗

PubMed1995

Nucleocapsid protein effects on the specificity of retrovirus RNA encapsidation.

We have analyzed the roles of Gag protein nucleocapsid (NC) domains in the packaging or encapsidation of retroviral RNAs into virus particles. We found that mutation of both zinc finger motifs of the human immunodeficiency virus (HIV) NC domain reduced but did not eliminate encapsidation of the HIV viral RNA. However, the NC mutations also resulted in a three- to fourfold reduction in the specificity of RNA encapsidation, as determined by comparison of virus-associated genomic and spliced RNA levels. As a complementary approach, we replaced the NC domain of Moloney murine leukemia virus (M-MuLV) with that of HIV. Chimeric virus particles assembled efficiently, were of wild-type M-MuLV density, and cross-linked at NC cysteines. In encapsidation studies, wild-type M-MuLV precursor Gag (PrGag) proteins packaged M-MuLV transcripts more efficiently than HIV RNAs. In contrast, chimeric PrGag proteins possessing the HIV-1 NC domain in the context of the M-MuLV MA (matrix), p12, and CA (capsid) domains encapsidated HIV transcripts to a greater extent than M-MuLV transcripts. Our results support the notion that retroviral NC domains contribute toward both the efficiency and specificity of viral genomic RNA packaging.

Animals↗