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R B Luftig

Publications and source records attributed to R B Luftig.

At least 55 records · Page 3Linked to original sources
PubMed1987

Differences in the pI heterogeneity of virion and intracellular Moloney murine leukaemia virus p30s.

At least three different p30 forms which vary in isoelectric point (pI) were previously shown by two-dimensional (2D) gel electrophoresis to be present in purified virions obtained from several strains of murine leukaemia virus (MLV). This heterogeneity which had been identified by Coomassie Brilliant Blue staining has been further characterized by immunological techniques. Using as substrates two Moloney (M) MLV chronically infected cell lines (MJD-54 and clone 2 cells), we found that (i) all p30s had the antigenicity of M-MLV p30, when analysed by immunoblotting of virion proteins with anti-p30 sera, and (ii) when cells were labelled with [35S]methionine, a 14C-amino acid mixture, or [14C]serine and lysates of purified virions were immunoprecipitated with goat anti-p30 sera, four p30 spots (pI 6.0, 6.1, 6.3 and 6.6) could be clearly identified. These results strongly support the viral origin of the heterogeneous p30 spots. We next examined infected cell lysates in an attempt to pinpoint the molecular basis of this heterogeneity. When we immunoprecipitated p30s from labelled cell lysates utilizing goat anti-p30 sera it was observed that in contrast to the four virion p30s, there were only three intracellular p30s (pI 6.0, 6.3 and 6.6), there was a threefold greater amount of the intracellular compared to the virion form of p30 with pI 6.0, tryptic peptide maps of both virion and intracellular p30s labelled either with [35S]methionine or 125I showed basically similar patterns with only slight differences in intensity among certain peptides for the p30s with pI 6.1, 6.3 and 6.6, and the intracellular p30 with pI 6.0 had a peptide that was not present in any of the other p30s. These results suggest that due to some as yet uncharacterized modification(s) of p30 and/or some structural differences between different p30s, a heterogeneity in pI exists. This may be important for assembly of the virion capsid. However, it is also possible that the p30 heterogeneity reflects the presence of multiple M-MLV proviruses within each of the infected cell clones.

Antibodies, Viral↗
PubMed1987

Characterization of a conformationally sensitive epitope on Moloney murine leukemia virus gp70.

We describe here several properties of a conformationally sensitive epitope common to the Moloney (M) and Rauscher (R) murine leukemia virus (MLV) gp70 family of glycoproteins, e.g., M-MLV gp70 and R-MLV gp71. This epitope was not detected by western blotting or by enzyme-linked immunosorbent assay experiments with three different lots of polyclonal R-MLV gp69/71 sera. However, it was detected when a specific monoclonal antibody, R47, was used in western blotting or immuno-dot-blotting experiments with the two viruses. R47 maps to a central 14-kd segment on R-MLV gp71 which spans an important structural domain, namely, that corresponding to the recombination site between ecotropic and endogenous envelope glycoprotein-coding sequences. Analysis of the western as well as dot blots developed with the R47 monoclonal antibody showed that about a 25-fold higher affinity for the epitope existed on R-MLV gp71, relative to M-MLV gp70. It thus appears that R-MLV and M-MLV gp70, although very closely related both structurally and serologically, are conformationally distinct in one domain, namely, the site of recombination between ecotropic and endogenous gp70-coding sequences.

Amino Acid Sequence↗
PubMed1986

Inhibition of cleavage of Moloney murine leukemia virus gag and env coded precursor polyproteins by cerulenin.

Cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, has been shown to significantly decrease (greater than 75%) the amount of Moloney murine leukemia virus (MMuLV) released into the culture medium of chronically infected mouse fibroblasts (I. Katoh, Y. Yoshinaka, and R.B. Luftig, 1986, Virus Res., in press). In order to clarify the mechanism by which this decrease in virus production occurs, we analyzed the kinetics of gag and env coded protein synthesis in M-MuLV infected, cerulenin-treated cells by immunoprecipitation with monospecific antisera to p30, p12, p10, gp70, and p15(E). We found that in pulse (15 min-2 hr)-chase (0-4 hr) experiments the cleavage of not only Pr65gag to p30 and other gag coded proteins but Pr80env to gp70 and Pr15(E) as well, was greatly reduced by cerulenin treatment. Further, since the total amount of label in the Pr65gag and Pr80env bands remained about the same or was slightly decreased in 2-hr pulsed, cerulenin-treated cells, this suggests that cerulenin decreases virus production, in part, by inhibiting the cleavage of both precursor gag and env coded polyproteins during virus assembly and budding at the cell membrane. We also observed that at longer chase periods (4 hr), the effect of cerulenin could be partially overriden in that minor amounts of cleaved gag and env coded polyproteins were produced and assembled into virion particles. However, these particles contained abnormally large amounts of the uncleaved precursor Pr65gag, suggesting that maturation was incomplete. The above results suggest two independent, but not exclusive, possible mechanisms of cerulenin action to block M-MuLV production, viz. cerulenin decreases the pool of fatty acids, thereby inhibiting fatty acid acylation of Pr65gag, as well as Pr80env, and thus preventing the interaction between gag (the p15 antigenic determinant on Pr65gag) and env [the p15(E) antigenic determinant of Pr15(E)] coded gene products at the cell membrane needed for efficient virus assembly (M. Satake and R. B. Luftig, 1983, Virology 124, 259-273), and cerulenin inhibits one or more proteolytic enzymes responsible for the cleavage of Pr65gag and Pr80env.

Antifungal Agents↗
PubMed1986

The effect of cerulenin on the synthesis of the precursor gag polyprotein in defective murine leukemia and sarcoma virus producing cell lines.

The effect of cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, on the synthesis of the precursor gag polyprotein, Pr65gag in a defective murine leukemia virus (334C) producing murine cell line (3JE) and a defective murine sarcoma virus (Gazdar) producing hamster cell line (HTG-2) was examined. In contrast to Moloney murine leukemia virus (M-MuLV) producing cell lines (MJD-54, clone 2) the amount of the Pr65gag remaining in the presence of cerulenin (20 micrograms/ml) was greatly reduced in both defective virus-infected cells. This effect appears specific for the Pr65gag polyprotein, since the env precursor polyprotein Pr80env was normally synthesized and remained undegraded in cerulenin-treated 3JE-infected cells. Thin-section electron micrographs showed an increased accumulation of virion particles in vesicles of treated HTG-2 cells.

Antifungal Agents↗
PubMed1986

The effect of cerulenin on Moloney murine leukemia virus morphogenesis.

Cerulenin is an antibiotic that interferes with fatty acid synthesis in eukaryotic cells. It had been shown by Schultz and Oroszlan (1983), that murine leukemia virus (MuLV) Pr65gag, the polyprotein precursor to the virion core proteins contains the fatty acid myristate at its NH2 terminus. We showed that when 20 micrograms/ml of cerulenin is added for 3 h to mouse fibroblasts chronically infected with Moloney (M)-MuLV it causes a greater than 4-fold decrease in virus production. This is accompanied by an accumulation of uncleaved Pr65gag in the infected cells. Further, thin-section electron micrographs of cerulenin-treated cells show a 2-fold increase in the number of nascent-budding forms, as well as the appearance of aberrant viral forms at the cell membrane. This suggests that the failure to add myristic acid to Pr65gag prevents their proper assembly into viral particles.

Animals↗
PubMed1986

Antigenic differences among multiply charged Moloney murine leukemia virus p30 polypeptides found inside infected cells.

At least three Moloney murine leukemia virus (M-MuLV) p30 polypeptides (p30's), viz., a major species at pI 6.3 and two minor ones at pI 6.1 and pI 6.6, have previously been identified in purified virions by 2-dimensional gel electrophoresis and chromatofocusing (Katoh, I., Yoshinaka, Y. and Luftig, R.B. (1984) J. Gen. Virol. 65, 733-741). We have observed a similar, but distinctive pI pattern for [35S]methionine-labeled MuLV p30's in lysates from chronically infected (MuLV) cells. The variation in pI pattern of the intracellular MuLV p30's was dependent on the type of p30 reactive antibody used for immunoprecipitation. Specifically: a p30 spot with pI 6.3 was always precipitated as the major spot with three different antibodies, minor spots with pI 6.0 and 6.6 were variably seen dependent on the antibody used, and an intracellular p30 spot at pI 6.1 was only precipitated with a rat p30 monoclonal antibody but not with monospecific mouse or intact MuLV cross-reacting p30 sera. These results indicate that first, there are differences between the pI pattern of virion and intracellular MuLV p30's, and second, the antigenic determinants of intracellular p30's vary dependent on the antibody used for immunoprecipitation.

Animals↗
PubMed1986

Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus.

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

Acylation↗
PubMed1985

Evidence for the existence of a thermolabile element in adenovirus type 5 that plays a role during an early stage of infection.

In an attempt to learn the molecular mechanism behind the initial steps (before genome expression) of adenovirus infection, the effect of temperature treatment of adenovirus type 5 particles on their infectivity was studied. It was found that adenovirus type 5 could be inactivated by temperature treatment at between 43 and 44 degrees C. Both biochemical and biophysical examination of adenovirus particles failed to disclose any significant changes after exposure to temperatures up to 45 degrees C. Further, viral particles treated with the same temperature range retained their ability to attach to cell surface receptors and to penetrate the plasma membrane. Application of a temperature pulse of 44 degrees C to adenovirus-infected HeLa cells indicates that the infection can be inhibited by exposure of infected cells to a brief temperature pulse of 44 degrees C between 10 and 50 min postadsorption with a mid-point of 30 min postadsorption. In addition, electron microscopic examination of HeLa cells following a temperature pulse of 44 degrees C suggests that there is a failure to accumulate viral particles at the perinuclear region. These findings support the hypothesis that adenovirus contains a thermolabile element which plays a crucial role during an early stage of infection. Possible correlation of this element with the adenovirus endogenous protein kinase is also examined and discussed.

Adenoviridae Infections↗
PubMed1985

An unexpected effect of divalent cations on the adenovirus endogenous protein kinase: alteration in the specificity of phosphorylation.

The adenovirus endogenous protein kinase specifically phosphorylates capsid protein IIIa in the presence of Mg2+, utilizing either ATP or GTP as a phosphate donor. When Mn2+ is substituted for Mg2+, in the presence of ATP, phosphorylation of IIIa is enhanced by 2-3 fold. However, in addition to IIIa phosphorylation, the core proteins V and VII are now phosphorylated. A similar finding is made when Co2+ is used instead of Mg2+. Further, when Mn2+ or Co2+ is substituted for Mg2+, the phosphoamino acid residue profile is changed, viz., instead of only phosphoserine being labeled, phosphothreonine is now extensively labeled. These results are specific for the above endogenous viral proteins, since when the viral protein kinase is used to phosphorylate added exogenous substrates such as casein, no enhancement of phosphorylation nor any change in phosphoamino acid profile is achieved by substituting Mn2+ for Mg2+. It is thus likely that MnATP or CoATP somehow interacts differently with adenovirus structural proteins than does MgATP and this facilitates their accessibility to the enzyme.

Adenosine Triphosphate↗
PubMed1985

In vitro cleavage of Pr65gag by the Moloney murine leukaemia virus proteolytic activity yields p30 whose NH2-terminal sequence is identical to virion p30.

In vitro cleavage of Gazdar murine sarcoma virus Pr65gag, which has all of the antigenic determinants of Moloney murine leukaemia virus Pr65gag, i.e. p15, p12, p30 and p10, by the Moloney murine leukaemia virus proteolytic activity yielded a p30 whose partial NH2-terminal sequence was identical to Moloney murine leukaemia virus. Both [3H]leucine-labelled and unlabelled Pr65gag were used to generate the cleaved p30.

Amino Acid Sequence↗
PubMed1985

Evidence for the ubiquitous presence of a protein kinase in human adenoviruses capable of preferentially phosphorylating capsid protein IIIa.

Several years ago a protein kinase activity was demonstrated in subgroup C adenoviruses. In this report we have demonstrated the presence of a similar protein kinase activity in adenoviruses of subgroups A, B, and D; like the subgroup C adenovirus kinase, it preferentially phosphorylates capsid protein IIIa. Further, we have shown that the protein kinase activity is independent of the host cell line used to grow the virus and is not inhibited by N-tosyl-L-lysine chloromethyl ketone or by N-tosyl-L-phenylalanine chloromethyl ketone (known inhibitors of other protein kinases). These results suggest that the adenovirus-associated protein kinase is an inherent component of all mature adenovirus particles, and as such may play a role in the virus life cycle.

Adenoviruses, Human↗
PubMed1984

Murine retrovirus Pr65gag forms a 130K dimer in the absence of disulfide reducing agents.

Gazdar-murine sarcoma virus (Gz-MSV) particles, obtained from tissue culture fluids of chronically infected HTG-2 hamster cells are immature in morphology and contain uncleaved Pr65gag as the predominant protein (greater than 95% Coomassie blue stain) (A. Pinter and E. deHarven, 1979, Virology 99, 103-110; Y. Yoshinaka and R. B. Luftig, 1982, Virology 118, 380-388). When Gz-MSV particles are disrupted in 1% sodium dodecyl sulfate (SDS) and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) in the absence of reducing agents, such as beta-mercaptoethanol (beta-MSH) almost half of the Pr65gag Coomassie blue-stained band is detected as a band at a Mr of 130K. Electrophoretic blotting studies with monospecific antisera against MuLV p30, p15, p12, and p10 showed that the 130K band cross-reacted with all four antigens suggesting that it was a dimer of Pr65gag. Two-dimensional (2D) SDS-PAGE where the first dimension was run under nonreducing conditions and the second with beta-MSH, supported the contention that the 130K band was a dimeric complex of Pr65gag. One also saw minor amounts of a 260K and higher polymeric forms of Pr65gag on the SDS gels, suggesting that polymeric forms may exist as well. When 32P-labeled Gz-MSV particles obtained by in vivo labeling of infected HTG-2 cells with [32P]PPi were electrophoresed on SDS-PAGE, only 10% of the 32P label was detected at the 130K position. In contrast, 30% of the Coomassie blue-stained Pr65gag material was found at 130K on the 2D gels. This suggests that unphosphorylated Pr65gag is more likely to participate in dimer formation than phosphorylated Pr65gag. Pr65gag of Moloney murine leukemia virus (M-MuLV), which is present as a minor (5% of stain) protein band on SDS-PAGE also showed 130K dimers. Further, in beta-MSH-deficient SDS preparations of Gz-MSV, electrophoresed after trypsin treatment, a 32K band that stained with p15, but not p10, p12, nor p30, antisera was observed. If beta-MSH was added, this band was no longer present. Thus Pr65gag dimerization in immature MuLV particles appears to at least involve the p15 region of the polyprotein. Since p15 is an extremely hydrophobic protein, formation of Pr65gag dimers may occur when virion precursor proteins are brought to the cell membrane during virus assembly.

Animals↗
PubMed1984

Murine leukaemia virus p30 heterogeneity as revealed by two-dimensional gel electrophoresis is not an artefact of the technique.

We have utilized two-dimensional (2D) gel electrophoresis [the first dimension being a linear pH gradient (5 to 8) and the second and 8 to 15% acrylamide gradient] to characterize the virion protein, p30, from several strains of purified murine leukaemia virus (MuLV). In all cases, we found that there was a predominant (70 to 90%) Coomassie Brilliant Blue-staining p30 spot, as well as several other species which differed in pI. The major p30 spot differed in pI among different MuLV strains and the minor spots varied depending on the host cell used to grow the virus. Specifically, (i) Moloney (M)-MuLV/NIH-3T3 showed two spots, a major one at pI 6.3 and a more acidic one, (ii) AKR/NIH-3T3, AKR/mouse embryo, and Gross/NIH-3T3 showed four spots, with the two basic, minor spots of AKR/NIH-3T3 appearing relatively decreased in intensity, and (iii) Rauscher (R)-MuLV/JLS-V9 (BALB/c) showed two spots, a major one with greater than 90% of the estimated Coomassie Brilliant Blue stain at a pI of 6.5 and a minor, acidic one. The major spots of AKR and M-MuLV viruses also differed in pI. The major spot of the AKR and Gross N-tropic viruses had a pI of 6.7 while that of NB-tropic virus M-MuLV had a pI of 6.3. The possibility that the heterogeneity observed in p30 was an artefact of the 2D gel technique had to be considered since urea was used to denature proteins in the first dimension of the gel. This possibility was made unlikely by our finding that another technique, chromatofocusing, gave the same results. Specifically, M-MuLV/JLS-V9 p30, when separated on chromatofocusing columns under non-denaturing conditions yielded three peaks, each of which directly corresponded to the three spots (pI: 6.1, 6.3, 6.6) observed on 2D gels. Furthermore, tryptic peptide maps of the major (pI 6.3) and one of the minor (pI 6.6) M-MuLV spots, although very similar in peptide composition, showed about five clearly defined differences. These results indicate (i) that the p30s of several N- and NB-tropic viruses are heterogeneous in pI, and (ii) for one particular MuLV, the p30 heterogeneity can be explained by a difference in amino acid composition. These findings of p30 charge heterogeneity may reflect either the presence of several different p30s in each virus particle and/or a heterogeneity in the virus population.

AKR murine leukemia virus↗
PubMed1983

Comparative immunofluorescence of murine leukemia virus-derived membrane-associated antigens.

We recently reported that the distribution and location of Moloney murine leukemia virus-derived membrane-associated gp70 and p30 antigens on the surface of 3.7% formaldehyde-fixed, chronically infected mouse fibroblasts were completely distinct, as judged by immunofluorescent light microscopy (M. Satake, P. N. McMillan, and R. B. Luftig (1981), Proc. Nat. Acad. Sci. USA 78, 6266). gp70, one of the two env gene products, exhibited a multiple-dot fluorescent pattern on the external surface of infected cells, while p30, one of the gag gene products, exhibited a diffuse fluorescence pattern which was apparently derived from Pr65gag molecules associated with the cytoplasmic face of the cell membrane. We have now examined the membrane fluorescence patterns of p15E, the other env gene product, as well as p15, p12, and p10, the other gag gene products. In these studies, both multivalent and monoclonal antibodies as well as fluorescein- and rhodamine-conjugated probes were used. We found that: (i) each of the env gene products, gp70 and p15E, exhibited characteristic and distinctive multiple-dot staining patterns. Further, each protein was labeled on intact cells with 125I-protein A plus homologous antiserum, confirming that both gp70 and p15E had externally exposed antigenic determinants. (ii) Among the gag gene products, p15 exhibited a different membrane fluorescence pattern than the diffuse pattern seen with p30, p12, and p10. The p15 pattern had an additional multiple-dot component. (iii) By double immunofluorescence we observed that the p15E and p15 multiple-dot patterns were superimposable at the same loci on infected cells. These three results suggest, first, that the cleavage of gp70 and p15E occurs prior to the arrival of the env polyprotein precursor at the cell surface and, second, there is an association between p15E and p15 antigenic determinants at the cell membrane. This latter association between an env and a gag gene product may be important for viral assembly.

Animals↗
PubMed1983

The cytoskeleton of murine leukemia virus (MuLV)-infected mouse fibroblasts as observed under varying conditions of formaldehyde fixation.

Mouse fibroblasts chronically infected with Moloney murine leukemia virus (MuLV) were fixed using variable amounts of formaldehyde, then examined by indirect immunofluorescence light microscopy. Several antisera were employed to detect both external and internal antigens associated with the cells, eg, MuLV gp70, tubulin, vimentin, and actin. Our results indicate that the cell membranes could be partially permeabilized to IgG molecules directed against the three cytoskeletal antigens only after 3.7%, but not 1%, formaldehyde treatment. Complete permeabilization was achieved by subsequent acetone treatment of cells after 3.7% formaldehyde fixation. In such cells, normal-appearing cytoskeletal networks of microtubules and intermediate filaments were observed. Stress fibers were also seen; however, they appeared less numerous and thinner than those of uninfected mouse fibroblasts. Further, a significant amounts of F-actin fluorescence was localized in granules in the cytoplasm of infected cells. Similar observations were made using JLS-V9 mouse cells chronically infected with 334C virus, another MuLV. These results taken together suggest that subtle differences exist in the organization of actin within MuLV-infected and uninfected mouse fibroblasts.

Actins↗
PubMed1983

The adenovirus type 5 capsid protein IIIa is phosphorylated during an early stage of infection of HeLa cells.

[32P]Orthophosphate-preincubated and thus 32P-labeled ATP-containing HeLa cells were infected with adenovirus type 5 particles. Following a 30 min infection period, viral particles were recovered and purified from infected cells by means of sucrose gradient and CsCl equilibrium centrifugation. Analysis of such recovered adenovirus particles by gel electrophoresis revealed that the capsid protein IIIa was phosphorylated. Further, a close resemblance was found in the tryptic peptide map of this in vivo phosphorylated IIIa and that of in vitro phosphorylated IIIa obtained from dialyzed virions. These results and other observations suggest that the previously known in vitro phosphorylation of IIIa with experimentally produced penton-free adenovirus particles likely mimics a phenomenon which takes place at an early stage of infection.

Adenosine Triphosphate↗
PubMed1983

Separation of a murine leukaemia virus protein kinase activity from its Pr65gag polyprotein substrate after DNA--cellulose chromatography.

We have recently found that, in vitro, the murine leukaemia virus (MuLV)-associated protein kinase activity predominantly phosphorylates Pr65gag, a virus protein present in relatively small amounts in partially purified virus preparations. Other virus proteins, such as p10, Pr27gag and Pr40gag, are also phosphorylated in vitro, but to a lesser degree. Furthermore, when immature core subparticles which are enriched in Pr65gag are prepared from virions by Sepharose 6B exclusion column chromatography, about 50% of the kinase activity (as assayed with the exogenous substrate phosvitin) remains associated with the cores. We report here that this core-associated activity is distinct from Pr65gag since it can be separated from Pr65gag by chromatography on denatured DNA--cellulose columns followed by centrifugation of the 0.2 M-NaCl-eluted fraction. Under these conditions, Pr65gag is pelleted while the kinase activity, which can phosphorylate both endogenous (MuLV Pr65gag and p10) as well as exogenous (phosvitin) substrates, remains in the supernatant. Interestingly, when the amount of Pr65gag is reduced, as in such preparations, p10 then becomes more heavily phosphorylated.

Chromatography↗