Gag precursors of HIV and SIV are cleaved into six proteins found in the mature virions

During retroviral maturation gag precursors are proteolytically cleaved to mature gag proteins. The number of mature gag proteins and their order in the gag precursors of HIV-I3B, SIVMne (captive macaque isolate), and SIVCat (wild mangabey isolate) has been determined by N-terminal amino acid sequence analysis of mature gag proteins and alignment with predicted sequences of homologous gag precursors. For HIV-1 and SIVs maturation proteolysis results in six gag proteins and the gag precursor cleavage pattern is distinctive and different from cleavage patterns for all other known retroviruses.     

Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate

At 37 degrees C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs greater than or equal to 3 mm). Irrespective of breed, genotype or follicle size, the mean (+/- s.e.m.) calculated association rate constant, (ka) was 7.3 (+/- 0.8) x 10(5) litres mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was less than 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (less than or equal to 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or greater than or equal to 3 mm). The overall mean +/- s.e.m. (n = 24) Bmax and Kd values were 16.7 +/- 0.8 fm/mg protein (i.e. approximately 800 available receptor binding sites/cell) and 1.1 +/- 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.     

Plasma clearance of radiolabelled IGF-1 in the late gestation ovine fetus

We investigated the distribution of radiolabelled IGF-1 in the late gestation ovine fetus by exclusion gel chromatography following intravenous injection of 125I rh (recombinant human) met-IGF-1 into the chronically instrumented fetal lamb (120-130 days, n = 7). One minute after injection of 125I rh met-IGF-1 into the fetal femoral vein, 20.9 +/- 3.1% of the counts circulated in the 150K binding protein region, 55.0 +/- 3.7% in the 50K binding protein region and 18.7 +/- 0.6% in the free or 7K region. The chromatographic profiles obtained in the fetus were in general similar to those previously seen in the adult sheep. After an initial equilibration phase the half life of IGF-1 associated with the 150K binding fractions were 412.1 +/- 103.6 min. Two phases of clearance were observed for IGF-1 in association with the 50K binding fractions, an initial phase with a half life of 30.6 +/- 4.5 min followed by a second phase with a half life of 202.3 +/- 10.3 min. The 7K or 'free' form of IGF-1 had an initial half life of 12.6 +/- 5.1 min. Chromatography of samples of fetal tracheal fluid, fetal urine, amniotic fluid, maternal uterine venous plasma and maternal systemic plasma showed no movement of intact IGF-1 out of the fetal circulation into the fetal fluids or into the maternal circulation. However, when simultaneous samples were obtained from the fetal femoral artery and umbilical vein, higher radioactivity was consistently observed in the fetal femoral artery raising the possibility of placental uptake of IGF-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of murine leukemia virus by compounds that react with the zinc finger in the viral nucleocapsid protein.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two copies of the conserved sequence motif C-X2-C-X4-H-X4-C. The conserved cysteine and histidine residues coordinate a zinc ion in each such motif. Rice et al. (W. G. Rice, J. G. Supko, L. Malspeis, R. W. Buckheit, Jr., D. Clanton, M. Bu, L. Graham, C. A. Schaeffer, J. A. Turpin, J. Domagala, R. Gogliotti, J. P. Bader, S. M. Halliday, L. Coren, R. C. Sowder II, L. 0. Arthur, and L. E. Henderson, Science 270:1194-1197, 1995) have described a series of compounds which inactivate human immunodeficiency virus type 1 (HIV-1) particles and oxidize the cysteine thiolates in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. The killed MuLV particles were found to be incapable of synthesizing full-length viral DNA upon infection of a new host cell. When MuLV particles are synthesized in the presence of one of these compounds, the normal maturational cleavage of the Gag polyprotein does not occur. The compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV- I by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses and the lethality of all known mutations altering the zinc-binding residues suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.     

Stopped-flow mechanistic study of bromide substitution by an organonitrile at an iron(II) phosphinic centre; a π-electron driven process

The kinetics of the displacement reactions of the bromide ligands of trans-[FeBr₂(depe)₂] (depe = Et₂PCH₂CH₂PEt₂) by the organonitrile NCCH₂C₆H₄OMe-4, in tetrahydrofuran (either in the absence or in the presence of added Br⁻), to give the corresponding mono- and dinitrile complexes trans-[FeBr(NCCH₂C₆H₄OMe-4)(depe)₂]⁺ and trans-[Fe(NCCH₂C₆H₄OMe-4)₂(depe)₂]²⁺, have been investigated by stopped-flow spectrophotometry. The substitution reaction occurs by a mechanism involving rate-limiting dissociation of bromo ligands to form the unsaturated intermediates [FeBr(depe)₂]⁺ (k₁ = 1.52 ± 0.02 s⁻¹) and [Fe(NCR)(depe)₂]²⁺ (k₃ = 0.063 ± 0.008 s⁻¹) which add the nitrile ligand to form those nitrile complexes. The competition between the nitrile and Br⁻ for such metal centres has also been investigated and a stronger inhibiting effect of added Br⁻ is observed for the substitution of the second bromo ligand relative to the first one. The kinetic data are rationalized in terms of π-electronic effects of these unsaturated metal centres and of the bromide and nitrile ligands.     

Scanning force microscopy reveals ellipsoid shape of chicken erythrocyte nucleosomes.

Scanning force microscopy was used to investigate the conformation of hypotonic spread chicken erythrocyte nucleosomes. Nucleosomal chains were prepared in low-salt conditions and fixed before centrifugation onto glass coverslips and air drying. The images of single nucleosomes were isolated by image processing, and the height and geometry of the resulting three-dimensional structures were investigated. An average nucleosome height of 4.2 +/- 1.1 nm was determined. A virtual cross section at half-maximum height of the nucleosome structure was used for a characterization of the nucleosome geometry. The shape of this cross section was best described by an ellipse with an aspect ratio (major/minor axis) of approximately 1.30.     

Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA.

The mus209B1 mutant of Drosophila melanogaster exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Our discovery that mus209 encodes proliferating cell nuclear antigen (PCNA), which is an indispensable component of the DNA replication apparatus, suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification.     

An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.