Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

Vesicular stomatitis virus (VSV) particles formed at early times after infection contain only one-third the amount of viral glycoportein (G protein), relative to the major internal structural proteins M and N, as is found in particles released later. These "early" particles also have a lower density in equilibrium sucrose gradients than do those formed later; however, the sedimentation velocity and specific infectivity of these two classes of particles are the same. VSV-infected cells also release virus-like particles which sediment considerably faster than authentic virions and contain a higher-than-normal proportion of the VSV G protein relative to internal VSV proteins. These particles have a reduced specific infectivity but a normal density in sucrose gradients. All classes of VSV virions contain a constant proportion of M and N polypeptides. The ratio of G protein to M or N protein, in contrast, can vary over a sixfold range; this implies that an interaction between a precise number of surface G proteins with either of the underlying M and N proteins is not a prerequisite for budding of infectious viral particles from the cell surface.     

Role of heterologous and homologous glycoproteins in phenotypic mixing between Sendai virus and vesicular stomatitis virus.

Phenotypic mixing between Sendai virus and vesicular stomatitis virus (VSV) or the mutant VSV ts045 was studied. Conditions were optimized for double infection, as shown by immunofluorescence microscopy. Virions from double-infected cells were separated by sequential velocity and isopycnic gradient centrifugations. Two types of particles with mixed protein compositions were found. One type was VSV particles with Sendai virus spikes, i.e., phenotypically mixed particles. A second type was Sendai virus-VSV associations, which in plaque assays also behaved as phenotypically mixed particles. The ratio of VSV G protein to Sendai virus glycoproteins on the cell surface was varied, using the VSV mutant ts045 in double infections. Thus, different amounts of the VSV G protein were allowed to reach the cell surface at 32, 38, and 39 degrees C in Sendai virus-infected cells. However, a fixed number of Sendai virus spikes was always found in the ts045 virions. This represented 12 to 16% of the number of G proteins present in normal VSV. Furthermore, the yield of ts045 virions was radically reduced during double infection when the temperature was raised to block G-protein transport to the cell surface, suggesting that the Sendai virus glycoproteins were not able to compensate for G protein in budding. These results emphasize the role of the G protein in VSV assembly.     

Delayed formation of defective interfering particles in vesicular stomatitis virus-infected cells: kinetic studies of viral protein and RNA synthesis during autointerference.

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.     

Effect of altered G levels on deposition of particulates in the human respiratory tract.

The primary mode of depositon of particles in the respiratory tract in the size range 0.5-10 mum diam (unit density) is sedimentation. The rate of sedimentation is directly proportional to the velocity of settling of the particle. Therefore, the total deposition of particles in the respiratory tract as well as the region of deposition is affected by changes in gravity. Human subjects were exposed to aerosols of 2.02-mum-diam polyvinyltoluene particles at 0, 0.5, 1.0, and 2.0 G. Total deposition was measured at each G level. Results indicate an almost linear increase in total deposition with increasing G levels over the range studied. The deposition measured at 1 G was less than reported in earlier experiments and the deposition at levels less than 1 G was less than had been calculated by Muir and Beeckmans. These data show that although sedimentation plays the major role in depostion of 2.02 mum particles, it is less than previously described.     

Cell cycle dependent G2 delay and killing of L929 cells after exposure to 241Am alpha particles

Survival and G2 delay of L929 mouse fibroblasts exposed to 3.4-MeV alpha particles depend on the cell age at the time of irradiation. Greatest sensitivity for both endpoints has been found at the G1/S transition: The surviving fraction of G1/S cells is reduced to 0.11 following 1 Gy of alpha particles compared to 0.31 for early G1 cells. The G2 + M transit time rises from 3 hr for control cells to 22 and 30 hr for cells irradiated with 0.3 Gy in G2 or at the G1/S boundary, respectively. Cells irradiated in early G1 do not show increased G2 + M transit times. Growth delay as calculated for the entire population increases linearly with dose by 23 hr/Gy of alpha particles.     

Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/− ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/−) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.     

Lipid-poor apolipoprotein A-I in Hep G2 cells: formation of lipid-rich particles by incubation with dimyristoylphosphatidylcholine

Apolipoprotein A-I is a major secretory product of the human hepatoma cell line, Hep G2; approx. 70% of apolipoprotein A-I was separated from the medium as lipid-poor apolipoprotein A-I in the d greater than 1.21 g/ml fraction while 30% was associated with high-density lipoproteins (HDL) of d 1.063-1.21 g/ml. The lipid-poor apolipoprotein A-I contains 50% proapolipoprotein A-I which is similar to the isoform distribution in Hep G2 preformed HDL. We tested the ability of lipid-poor apolipoprotein A-I from Hep G2 to form complexes with dimyristoylphosphatidylcholine (DMPC) vesicles at DMPC/apolipoprotein A-I molar ratios of 100:1 and 300:1. Lipid-poor apolipoprotein A-I was recovered in complex form while at a 300:1 ratio, 68.8 +/- 6.3% was recovered. On electron microscopy, the former complexes were small discs 16.9 nm +/- 4.5 S.D. in diameter while the latter were larger discs 21.4 +/- 4.4 nm diameter. Non-denaturing gradient gel electrophoresis of complexes formed at a 100:1 ratio had a peak in the region corresponding to 9.64 +/- 0.08 nm; these particles possessed two apolipoprotein A-I molecules. At the higher ratio, 300:1, two distinct complexes were identifiable, one which banded in the 9.7 nm region and the other in the 16.9-18.7 nm region. The former particles contained two molecules of apolipoprotein A-I and the latter, three molecules. This study demonstrates that lipid-poor apolipoprotein A-I which is rich in more basic isoforms forms discrete lipoprotein complexes similar to those formed by mature apolipoprotein A-I. It is further suggested that, under the appropriate conditions, precursor or nascent HDL may be assembled extracellularly.     

Insertion of the human immunodeficiency virus CD4 receptor into the envelope of vesicular stomatitis virus particles.

Enveloped virus particles carrying the human immunodeficiency virus (HIV) CD4 receptor may potentially be employed in a targeted antiviral approach. The mechanisms for efficient insertion and the requirements for the functionality of foreign glycoproteins within viral envelopes, however, have not been elucidated. Conditions for efficient insertion of foreign glycoproteins into the vesicular stomatitis virus (VSV) envelope were first established by inserting the wild-type envelope glycoprotein (G) of VSV expressed by a vaccinia virus recombinant. To determine whether the transmembrane and cytoplasmic portions of the VSV G protein were required for insertion of the HIV receptor, a chimeric CD4/G glycoprotein gene was constructed and a vaccinia virus recombinant which expresses the fused CD4/G gene was isolated. The chimeric CD4/G protein was functional as shown in a syncytium-forming assay in HeLa cells as demonstrated by coexpression with a vaccinia virus recombinant expressing the HIV envelope protein. The CD4/G protein was efficiently inserted into the envelope of VSV, and the virus particles retained their infectivity even after specific immunoprecipitation experiments with monoclonal anti-CD4 antibodies. Expression of the normal CD4 protein also led to insertion of the receptor into the envelope of VSV particles. The efficiency of CD4 insertion was similar to that of CD4/G, with approximately 60 molecules of CD4/G or CD4 per virus particle compared with 1,200 molecules of VSV G protein. Considering that (i) the amount of VSV G protein in the cell extract was fivefold higher than for either CD4 or CD4/G and (ii) VSV G protein is inserted as a trimer (CD4 is a monomer), the insertion of VSV G protein was not significantly preferred over CD4 or CD4/G, if at all. We conclude that the efficiency of CD4 or CD4/G insertion appears dependent on the concentration of the glycoprotein rather than on specific selection of these glycoproteins during viral assembly.     

Properties of lipid-apolipoprotein association products. Complexes of human apo AI and binary phospholipid mixtures

The products resulting from the association of human apo A-I with binary phospholipid mixtures of dimyristoyl phosphatidylcholine (DMPC) and either dipalmitoyl (DPPC) or distearoyl (DSPC) phosphatidylcholine have been isolated and characterized. Effective lipid . protein complex formation was found to occur at the onset temperature for melting of the gel state, and equal incorporation of both lipid components of the binary mixture was observed. Two sizes of products were obtained, one containing 2 A-I molecules per complex and the other containing 3; the proportions of these two products depended upon the initial phospholipid/protein ratio employed. these two product species were found to be resolvable by density gradient centrifugation as well as gel filtration, reflecting substantial differences in composition as well as size. The ratio of DMPC to DPPC or DSPC was the same in the isolated complexes as in the initial mixture, suggesting that th protein does not associate preferentially with the fluid phase lipid, but with lipid domains in which the components are randomly distributed. Electron microscopy of recombinant particles containing a 2:1 ratio (w/w) of DSPC to DMPC revealed stacks of discs whose thickness was proportionately greater than for discs containing DMPC alone. Also of significance was the finding that recombinant discs containing 3 A-I molecules possessed a diameter approximately 1.5 times larger than recombinant discs containing 2 A-I molecules. These data are consistent with the model for the recombinant particles described by Tall et al. (Tall, A.R., Small, D.M., Deckelbau, R.J., and Shipley, G.G. (1977) J. Biol. Chem. 252; 4701-4711), in which the phospholipid is found as a circular bilayer, the thickness of which is dependent upon the length of the acyl chain, and around which the protein is distributed as an annulus.     

Purification of B. megatherium Phage G and Evidence for a Muralytic Enzyme as an Integral Part of the Phage

The Purification Of B. megatherium G phage is described and it is shown that DEAE cellulose chromatography combined with conventional methods gave a phage preparation which was at least 95 per cent pure, and contained 2.16 µg nitrogen/10¹¹ infective particles. The phage particle weight in molecular weight units was 91 x 10⁶. The small amount of contaminating material appeared to represent phage "ghosts." An essentially 1:1 ratio of particles to infective units was found when data from electron microscopic counts or data from chemical analysis were related to phage infectivity. Comparison, by several methods, of the G phage and coliphage T2 shows that T2 is 2.6 times larger than G phage. The specific activity of the muralytic component obtained by disintegration of phage preparations with urea was unchanged by the purification indicating that the phage-"bound" muralytic activity is an integral part of the phage structure.     

The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes

In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium.     

The glycogen-binding subunit of protein phosphatase-1G from rabbit skeletal muscle. Further characterisation of its structure and glycogen-binding properties

The glycogen-bound form of protein phosphatase-1 (PP-1G) was previously purified as a heterodimer composed of a 37-kDa catalytic (C) subunit and a proteolytically sensitive 103-kDa glycogen-binding (G) subunit [Str?hlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295-303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161-kDa protein, and that the 103-kDa species (now termed G') is itself a product of proteolysis. A second phosphorylation site for cAMP-dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP-1 at a slow rate, whereas site 1 was resistant to autodephosphorylation. PP-1G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen-protein particles during washing with a variety of solvents. The PP-1G holoenzyme was released from glycogen-protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP-1G. Binding experiments with purified PP-1G and glycogen indicated a bimolecular process with Kapp values corresponding to about 8 nM glycogen and 4 nM PP-1G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high-affinity glycogen-binding domain on the G subunit, and indicate that a physiological concentrations of phosphatase and glycogen, PP-1G should be almost entirely bound to glycogen.     

Structure of thylakoids in cells of Rhodopseudomonas viridis as influenced by growth conditions

A vessel was constructed for growth of photosynthetic bacteria at defined light intensity, temperature and partial pressure of oxygen.Under growth conditions at light intensities larger than 1,000 lx, the particles exposed by freeze-fracturing of thylakoids are unordered.Under growth conditions at light intensities lower than 30 lx, the particles seen are hexagonally arranged. If the oxygen partial pressure is increased from 0 to 30 mm Hg while keeping the light intensity at 30 lx, the particles seen in the thylakoids are found to be unregularly arranged.The protein pattern of thylakoids isolated from bacteria grown either at 2,000 lx or at 30 lx revealed a constant ratio of reaction centre polypeptide to either of the membrane polypeptides of 8 kdalton apparent Mr and 12 kdalton apparent Mr.Dedicated to Prof. Dr. G. Drews on occasion of his 60th birthday     

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant.

We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.     

Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.     

Antibody-Dependent and -Independent Protection following Intranasal Immunization of Mice with Rotavirus Particles

The ability to elicit protective immune responses after intranasal immunization with rotavirus particles, either with or without the attenuated Escherichia coli heat-labile enterotoxin LT(R192G) as an adjuvant, was examined in the adult mouse model. BALB/c mice were administered one or two inoculations of psoralen/UV-inactivated, triple-layered (tl) or double-layered (dl) purified rotavirus particles. Four weeks after immunization, mice were challenged with the murine rotavirus strain EDIM, and the shedding of rotavirus antigen was quantified. Rotaviruses used for immunization included EDIM and heterotypic simian (RRV), bovine (WC3), and human (89-12) strains. tl EDIM stimulated both systemic and intestinal rotavirus antibody responses and complete protection with as little as one 1-microgram dose. Inclusion of LT(R192G) (10 micrograms) significantly increased rotavirus antibody responses and reduced antigen concentrations needed for full protection. Both dl EDIM and heterotypic dl and tl particles stimulated protection, but they did so less than tl EDIM at comparable concentrations, either with or without LT(R192G). When B-cell-deficient microMt mice were immunized with tl EDIM particles, protection was reduced to levels similar to those induced with dl EDIM and heterotypic particles in BALB/c mice. However, dl EDIM particles induced similar levels of protection in both mouse strains. The protection stimulated by tl or dl EDIM particles was not diminished by CD8 cell depletion prior to immunization in either strain of mice. These results indicate that tl EDIM induced immunity at least partially through responses to its outer capsid proteins, presumably by stimulation of serotype-specific neutralizing antibody. In contrast, the other particles stimulated protection primarily by an antibody-independent mechanism. Finally, depletion of CD8 cells had no effect on protection by either mechanism.     

Biological responses in known bystander cells relative to known microbeam-irradiated cells

Normal human fibroblasts in plateau phase ( congruent with 95% G(1) phase) were stained with the vital nuclear dye Hoechst 33342 (blue fluorescence) or the vital cytoplasmic dye Cell Tracker Orange (orange fluorescence) and plated at a ratio of 1:1. Only the blue-fluorescing nuclei were microbeam-irradiated with a defined number of 90 keV/microm alpha particles. The orange-fluorescing cells were then "bystanders", i.e. not themselves hit but adjacent to cells that were. Hit cells showed a fluence-dependent induction of micronuclei as well as delays in progression from G(1) to S phase. Known bystander cells also showed enhanced frequencies of micronuclei (intermediate between those seen in irradiated and control cells) and transient cell cycle delays. However, the induction of micronuclei in bystander cells did not appear to be dependent on the fluence of the particles delivered to the neighboring hit cells. These are the first studies in which the bystander effect has been visualized directly rather than inferred. They indicate that the phenomenon has a quantitative basis and imply that the target for radiation effects cannot be considered to be the individual cell.     

Magnetic nanostructured composites using alginates of different M/G ratios as polymeric matrix

Alginate extracted from Sargassum fluitans and Macrocystis pyrifera with different molecular weights and mannuronic/guluronic ratios, M/G, were used as gel matrixes in order to obtain magnetic nanostructured composites. Magnetic nanocrystalline particles of iron oxides were formed inside the alginate matrix by in situ alkaline oxidation of ferrous ions. The magnetic materials obtained were subjected to several oxidative cycles and the increment in iron content was determined after each cycle. X-ray diffraction, magnetometry and M?ssbauer spectroscopy were used to examine the materials. The high magnetic response, the absence of hysteresis, and the centered paramagnetic doublet in the M?ssbauer spectra indicate the presence of nanocrystalline particles with a superparamagnetic behavior. X-ray diffractograms show peaks that correspond to maghemite. After the first cycle, Sargassum had four times the magnetic response of Macrocystis, which had more than twice the M/G ratio.     

The budding mechanism of spikeless vesicular stomatitis virus particles.

Virus particles, lacking the spike G-glycoproteins, are produced during infection of Vero cells with the vesicular stomatitis virus mutant ts045 at the restrictive temperature 39.5 degrees C. At this temperature the mutated G proteins are blocked in their intracellular transport in the endoplasmic reticulum. We have studied the role of the G proteins in the formation of these spikeless virus particles. The results showed that the spikeless particles contain a full complement of membrane anchors, derived from the carboxy-terminal end of the G protein. Our observations suggest that virus particles are formed at the restrictive temperature with G protein which is later cleaved to produce spikeless particles. We suggest that this is due to a leak of G protein to the cell surface at 39.5 degrees C where budding then takes place, presumably driven by a G protein C-terminal tail--nucleocapsid interaction.