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R R Rueckert

Publications and source records attributed to R R Rueckert.

At least 55 records · Page 3Linked to original sources
PubMed1984

Plaque assay for black beetle virus.

A rapidly growing strain of virus was used to develop a reliable plaque assay for Black beetle virus on monolayers of cultured Drosophila cells. Cell density of the monolayer was critical for successful plaque formation. The dose-response curve for plaque formation was linear, supporting earlier proposals that both RNA segments of the split genome reside in the same particle. The method greatly facilitates isolation of reassortant and variant strains of virus.

Animals↗
PubMed1983

Crystallization of a common cold virus, human rhinovirus 14: "isomorphism" with poliovirus crystals.

Crystals of rhinovirus 14 have been grown reproducibly. They diffract x-rays to a resolution of at least 3.5 A. The orthorhombic crystal unit cell contains two virions, each situated on a crystallographic twofold axis. At less than 30-A resolution, the space group approximates to 1222 with the particles possessing 222 pseudo crystallographic symmetry. The crystals are "isomorphous" with type I polio crystals [Finch, J. T. & Klug, A. (1959) Nature (London) 183, 1709-1714; Hogle, J. M. (1982) J. Mol. Biol. 160, 663-668], suggesting some similarities of structure between enteroviruses and rhinoviruses.

Crystallization↗
PubMed1983

Autonomous replication and expression of RNA 1 from black beetle virus.

Black beetle virions contain two RNAs. The smaller one, RNA 2, has previously been shown to be a messenger for viral coat protein. It is shown here, by infecting sensitized Drosophila cells with the individually purified RNAs, that the larger one, RNA 1, carries the viral gene(s) required for RNA polymerase functions. RNA 2 was dispensible for synthesis of viral RNA 1 and subgenomic RNA 3 but was essential for synthesis of RNA 2 and virions. Cells infected with RNA 1 alone produced RNA 3 in proportions 10- to 20-fold greater than cells infected with virions. This overproduction of RNA 3 decreased with increasing proportions of RNA 2 in the infecting RNA 1. We conclude that RNA 1 is the previously unidentified progenitor of subgenomic RNA 3, whereas RNA 2 regulates the amount of RNA 3 produced in the infected cell.

Journal Article↗
PubMed1982

Evidence for intramolecular self-cleavage of picornaviral replicase precursors.

It has previously been shown that when encephalomyocarditis viral RNA is translated in cell-free extracts of rabbit reticulocytes, it synthesizes a virus-coded protease, p22, which is derived by cleavage of a precursor protein, C. Protein C is shown here to be cleaved by two different mechanisms, which were distinguished by their sensitivity to dilution. One mechanism was sensitive to dilution; the other was not. The biphasic cleavage behavior was unchanged by diluting incubation mixtures with untranslated reticulocyte extract instead of buffer, suggesting that both types of cleavage were mediated by virus translation products. It is proposed that the dilution-sensitive cleavage of protein C is due to a virus-coded protease, probably p22 itself, and that the dilution-independent cleavage is due to intramolecular self-cleavage of protein C.

Capsid↗
PubMed1982

Black beetle virus: messenger for protein B is a subgenomic viral RNA.

Black beetle virus induces the synthesis of three new proteins, protein A (molecular weight, 104,000), protein alpha (molecular weight, 47,000), and protein B (molecular weight, 10,000), in infected Drosophila cells. Two of these proteins, A and alpha, are known to be encoded by black beetle virus RNAs 1 and 2, respectively, extracted from virions. We found that RNA extracted from infected cells directed the synthesis of all three proteins when it was added to a cell-free protein-synthesizing system. When polysomal RNA was fractionated on a sucrose density gradient, the messengers for proteins A and alpha cosedimented with viral RNAs 1 (22S) and 2 (15S), respectively. However, the messenger for protein B was a 9S RNA (RNA 3) not found in purified virions. Like the synthesis of viral RNAs 1 and 2, intracellular synthesis of RNA 3 was not affected by the drug actinomycin D at concentrations which blocked synthesis of host cell RNA. This indicated that RNA 3 is a virus-specific subgenomic RNA and, therefore, that protein B is a virus-encoded protein.

Journal Article↗
PubMed1981

Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2.

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.

Journal Article↗
PubMed1980

Changes in three of the four coat proteins of oral polio vaccine strain derived from type 1 poliovirus.

Little is yet known about the nature, or extent, of the changes involved in attenuation of neurovirulent poliovirus. The tryptic comparison reported here, of coat proteins from the Sabin type 1 polio vaccine and parental Mahoney virus, provides a useful approach and affords some insight into this question. The main obstacle, separation of the labile proteins VP1 and VP2 in an intact state from the vaccine strain, was overcome by incorporating 3.5 M urea into an otherwise standard preparative gel electrophoresis system. Tryptic maps revealed six altered leucine-containing peaks: two in VP1, none in VP2, three in VP3, and one in VP4. It is estimated, after correcting for leucine-free peptides, that the coat protein sequences may have undergone some 10 to 13 amino acid replacements, roughly 1.5% of the total, in the course of attenuation leading to the vaccine strain.

Capsid↗
PubMed1980

Novel p19-related protein in Rous-associated virus type 61: implications for avian gag gene order.

Virions of Rous-associated virus type 61 contain a previously unrecognized p19-related protein, called p19f, which comigrates with gag protein p12 during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels but can be separated by gel filtration chromatography in 6 M guanidine hydrochloride. It is shown that the existence of p19f accounts for the earlier inability to order p27 and p12 by the pactamycin mapping procedure. Remapping with pactamycin by using methods which take this new protein into account yielded a gag gene order of NH2-p219-p27-p12-p15-COOH. It also confirmed earlier positions for the env and pol genes and placed unclassified protein p10 near a translational initiation site. The pactamycin-derived mapping position of p12 differs from reports based on tryptic analysis. An analysis of procedural shortcomings emphasizes the need for more definitive determinations of the avian gag gene order.

Avian Leukosis Virus↗
PubMed1980

Picornaviral VPg sequences are contained in the replicase precursor.

It has previously been shown that the RNA replicase of encephalomyocarditis virus contains two virus-coded proteins, D and E, which are produced in two successive proteolytic steps: (i) C leads to D + ?; and (ii) D leads to p22 + E. It is here shown (i) that virus protein H (molecular weight, 12,000) is the previously unidentified product of the first step and (ii) that VPg, a protein linked covalently to the virion RNA, yields two tryptic peptides found in protein C but not in protein D. The results suggest that VPg is derived by cleavage of protein C and that protein H may be intermediate. Preliminary experiments with VPg sequences in polioviral noncapsid protein 1b, the counterpart of encephalomyocarditis viral protein C, were inconclusive.

Encephalomyocarditis virus↗
PubMed1979

Translation of encephalomyocarditis virus RNA in reticulocyte lysates: kinetic analysis of the formation of virion proteins and a protein required for processing.

In cell-free extracts derived from rabbit reticulocytes, encephalomyocarditis RNA can be translated completely, and the products can be processed extensively to give encephalomyocarditis virion proteins and several nonvirion proteins, including a genome-coded protein required for processing. The latter is probably a protease. Translation is very efficient. Under typical conditions, each EMC RNA is translated approximately eight times during a 3-h period. Kinetic analyses (time-course experiments, pulse-chase experiments, and pulse-stop experiments) have been used to determine the time of appearance of major products, and these times have been correlated with map positions. The gene for the putative protease is located near the middle of the genome downstream from the virion protein genes. Ribosomes can travel the length of encephalomycarditis RNA within 30 min, but there is a delay in their progress along the RNA at some point soon after they traverse the region coding for virion protein precursors. This delay results in the accumulation of precursors for a period of about 10 min before the putative protease is made and virion proteins (epsilon, alpha, and gamma) are released by proteolysis.

Encephalomyocarditis virus↗
PubMed1979

Protease required for processing picornaviral coat protein resides in the viral replicase gene.

Partial purification of the encephalomyocarditis protease synthesized in extracts from rabbit reticulocytes shows that the activity responsible for cleaving coat precursor protein cosediments with a previously unmapped virus-coded protein with an apparent molecular weight of 20,000. Tryptic analysis shows that this protein is derived from protein D, a virus-coded component of the encephalomyocarditis RNA polymerase.

Capsid↗
PubMed1978

Proteins of Rous-associated virus 61, an avian retrovirus: common precursor for glycoproteins gp85 and gp35 and use of pactamycin to map translational order of proteins in the gag, pol, and env genes.

Cells infected by Rous-associated virus 61 (RAV-61) contained a precursor-like protein, pr90, that was specifically precipitated by antiserum directed against envelope glycoproteins, gp85 and gp35. Tryptic peptide mapping showed that pr90 contained tryptic sequences of both gp85 and gp35. Pactamycin mapping experiments indicated that the two glycoproteins are translated from the env-mRNA in the order (5') gp85--gp35. The pactamycin mapping experiments also indicated a translational order of p10--(p27, p12)--p15 for the gag proteins; this agreement with the order previously reported from tryptic mapping studies on precursor pr76 of avian myeloblastosis virus implied that the stoichiometry of the core proteins was unchanged when virions were assembled in the presence of pactamycin. The reverse transcriptase proteins, unlike those of the env and gag genes, fell on the right side of the pactamycin map. This result is in accord with the idea that most, if not all, of the reverse transcriptase protein is translated by read-through of the gag(pol) message rather than by translation of a hypothetical pol-mRNA devoted solely to synthesis of that protein.

Avian Leukosis Virus↗
PubMed1977

Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits.

Cytoplasmic extracts of rhinovirus 1A-infected HeLa cells, pulsed 15 min with [3H]leucine, contained a 13S subunit which was rich in the capsid precursor, peptide 92. After a 30-min chase, most of the capsid-related protein sedimented in a 14S peak that contained equimolar amounts of the capsid peptides epsilon, alpha, and gamma, and some residual chain 92. The 14S subunit could be dissociated at pH 4.8 into 6S subunits containing only epsilon, alpha, and gamma chains in equal proportions, indicating that the 14S subunit is an oligomer of (epsilon gamma alpha) protomers. These subunits resemble subunits previously identified in the assembly of enteroviruses. These observations support the idea that rhinovirus assembly is basically similar to that of enteroviruses. Comparative studies on the peptide stoichiometry of the virion and the capsid precursor subunits indicate that rhinovirus 1A can contain as many as 11 immature protomers per virion.

Capsid↗
PubMed1977

Proteins of Rous-associated virus type 61: polypeptide stoichiometry and evidence that glycoprotein gp35 is not a cleavage product of gp85.

The two glycoproteins, gp85 and gp35, of Rous-associated virus type 61 (RAV-61), were isolated from radiolabeled virions by gel electrophoresis and digested with trypsin. The chromatographic profile of the gp35 digest revealed no peaks in common with that of gp85; therefore, the smaller glycoprotein is not a cleavage product of gp85. The stoichiometry of radiolabeled RAV-61 proteins was studied by quantitative gel filtration and gel electrophoresis. Among the 11 polypeptides identified were 4 minor ones, including the beta(p91) and alpha(p64) chains of reverse transcriptase and two unidentified chains, p76 and p35; the latter two were unmasked by removing the virions' surface glycoproteins with a protease, bromelain. Virions contained some 15 to 30 molecules of reverse transcriptase.

Animals↗
PubMed1976

Evidence of ambiguous processing and selective degradation in the noncapsid proteins of rhinovirus 1A.

Pulse-chase kinetics and extensive pactamycin mapping studies show that the translation of rhinovirus 1A proceeds in the order: initiate-P1-S-P2-terminate, where P1 is the precursor to the capsid proteins, S is a stable primary gene product, and P2 is the precursor to a family of noncapsid products. Initial examination of the molar stoichiometry of the families of rhinoviral proteins in infected cells suggested that both the P1 and P2 regions were translated more frequently than the S region. However, we show that this apparent asymmetry in translation is an artifact arising from two phenomena: (i) ambiguous cleavage sites which result in two alternative products from the S region, having apparent molecular weights of 47,000 and 38,000, and (ii) several fates for the P2 precursors, including degradation of 35 to 45% of the P2 family to small unidentifiable products. Another artifact, a time-dependent shift in the pactamycin mapping position of polypeptide r-39, was traced to a selective inhibition of the rate of cleavage of its precursor (peak 76). The processing rate of the capsid precursor (peak 92) was not retarded by pactamycin.

Capsid↗