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Showing papers by "David Baltimore published in 1977"


Journal ArticleDOI
TL;DR: Digests of replicative intermediate RNA contained sufficient protein-pUp to suggest that this structure is at the 5'-end of most nascent poliovirion RNA molecules, suggesting that a protein-nucleotide structure acts as a primer for initiating synthesis of poliovirus RNA.
Abstract: The 5'-terminus of poliovirus polyribosomal RNA is pUp. A candidate for the 5'-terminus of poliovirion RNA was recovered as a compound migrating toward the cathode when 32P-labeled virion RNA was completely digested with ribonucleases T1, T2 and A and analyzed by paper ionophoresis at pH 3.5. Treatment with proteinase K reversed its direction of migration, indicating the presence of protein. Treatment with venom phosphodiesterase liberated all of the radioactivity as pUp, suggesting that poliovirion RNA has a protein-pUp 5'-terminus. Treatment of virion RNA with T1 ribonuclease alone generated a proteinase K-sensitive oligoribonucleotide. Analysis of the oligoribonucleotide using ribonucleases A and U2 showed its structure to be protein-pU-U-A-A-A-A-C-A-G. Digests of replicative intermediate RNA contained sufficient protein-pUp to suggest that this structure is at the 5'-end of most nascent poliovirus RNA molecules. We suggest that a protein-nucleotide structure acts as a primer for initiating synthesis of poliovirus RNA.

322 citations


Book ChapterDOI
01 Jan 1977
TL;DR: Defective interfering particles were discovered three decades ago by von Magnus using the influenza virus system and a great deal was learned from their physiological interactions with the host and from their interference with the multiplication of “infectious” standard virus.
Abstract: Defective interfering (DI) particles were discovered three decades ago by von Magnus (1947) using the influenza virus system. He called them “incomplete” or “immature” particles. Even though they could not be isolated or characterized biochemically, a great deal was learned about them from their physiological interactions with the host and from their interference with the multiplication of “infectious” standard virus (Gard and von Magnus, 1947; Bernkopf, 1950; von Magnus, 1951). Reviews by Henle (1950), by von Magnus (1954), and by Schlesinger (1959) on viral interference discuss the earlier work on this particular homologous interference caused by incomplete virus.

226 citations


Journal ArticleDOI
TL;DR: Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes.
Abstract: A template-dependent RNA polymerase has been isolated from poliovirus-infected cells by assaying for the ability of the enzyme to copy poly(A) complexed to an oligo(U) primer. The polymerase was solubilized with detergent, and RNA was removed by precipitation with 2 M LiCl. The solubilized polymerase required both poly(A) and oligo(U) for activity and was stimulated by Mg2+ but was inhibited by Mn2+. Poly(A)-oligo(U)-dependent poly(U) polymerase was not found in extracts of HeLa cells until about 2 hr after poliovirus infection, and then there was a linear increase in activity until about 5 hr. Analysis of the polymerase by glycerol gradient centrifugation showed that the majority of the activity sedimented at about 4 S, indicating that it was no longer complexed with high-molecular-weight RNA or cellular membranes. This poly(A)-oligo(U)-dependent polymerase activity could represent an important component of the poliovirus RNA-dependent RNA polymerase.

183 citations


Journal ArticleDOI
TL;DR: From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.
Abstract: Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. In contrast, the glycoprotein (G) was always membrane bound; it appeared to be directly inserted into membranes during its synthesis. Glycosylation of the G protein was completed only in smooth membrane fractions, possibly in the Golgi apparatus. After a minimum time of 15 min following its synthesis, G protein was incorporated into the surface plasma membrane, from which it was slowly shed into virions. These multiple processing steps probably account for its delayed appearance in virus. From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.

182 citations


Journal ArticleDOI
TL;DR: Results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane, and G protein behaved as in cells infected by mutants in N protein.
Abstract: Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation The movement of G, M, and N proteins to the virion bud appeared to be interdependent Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane

151 citations


Journal ArticleDOI
TL;DR: The small RNAs of the Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis as mentioned in this paper.
Abstract: The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro.

125 citations


Journal ArticleDOI
TL;DR: Two cell-associated forms of the glycoprotein (G) of vesicular stomatitis virus, termed G1 and G2, have been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that partial clycosylation of G occurs concomitantly with or immediately after its synthesis.
Abstract: Two cell-associated forms of the glycoprotein (G) of vesicular stomatitis virus, termed G_1 and G_2, have been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. G_1 has the higher electrophoretic mobility, but both forms migrate more slowly than G protein synthesized in a wheat germ cell-free system (G_0), which presumably is the unglycosylated form. G_1 is a kinetic precursor of the G_2 form, and the apparent cause of the electrophoretic difference between the two species is the presence of N-acetylneuraminic acid on the G_2 form. Conversion of G_1 to G_2 occurs 10 to 20 min prior to the appearance of the G_2 form of the protein on the cell surface. This suggests that the G protein may be completely glycosylated several minutes prior to its migration to the cell surface and that glycosylation is not the limiting step in its maturation. No glycoprotein comigrating with G_0 can be detected in the infected cells, even after 5-min labeling periods; this suggests that partial clycosylation of G occurs concomitantly with or immediately after its synthesis.

124 citations


Journal ArticleDOI
TL;DR: It is concluded that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules.
Abstract: Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3' and 5' ends of linear molecules.

112 citations


Journal ArticleDOI
TL;DR: Three of the mutants previously isolated in this laboratory, tsM502(V), tsM601( VI), and tsM602(VI), were shown to be defective in viral RNA synthesis at the nonpermissive temperature.
Abstract: The metabolism of viral RNA and proteins has been studied in cells infected with temperature-sensitive mutant strains of vesicular stomatitis virus. Certain viral proteins encoded by the mutant strains, usually the putative mutant protein for the assigned complementation group, were shown to be degraded more rapidly at the nonpermissive temperature than were the wild-type proteins. Group III mutants (tsG33, tsM301) encode M proteins which are degraded three- to fourfold faster than the wild-type protein. This defect cannot be fully rescued by coinfection with wild-type virus, and thus the defect appears to be in the M protein itself. Mutants tsM601 (VI) and tsG41(IV) encode N proteins which are degraded much faster than the wild-type protein and also share the property of being defective in replication of viral RNA, suggesting a correlation between these phenotypic properties. Furthermore, the L proteins of tsG11(I) and tsG13(I) are more labile than the wild-type protein at the nonpermissive temperature. The G protein of tsM501(V) did not undergo the change in electrophoretic mobility previously shown to be the result of sialylation, suggesting that it is defective in maturation or glycosylation at the nonpermissive temperature. Three of the mutants previously isolated in this laboratory, tsM502(V), tsM601(VI), and tsM602(VI), were shown to be defective in viral RNA synthesis at the nonpermissive temperature. Mutant tsM601(VI) was defective mainly in viral RNA replication, whereas tsM502(V) appeared to be totally defective for viral RNA transcription and replication at the nonpermissive temperature.

82 citations


Journal ArticleDOI
01 Oct 1977-Virology
TL;DR: Intact HeLa cells can bind virions of poliovirus type 1 and can subsequently convert them to altered particles by incubation at 37°, and with this simple assay procedure, quantitative parameters of binding and alteration can be measured.

81 citations


Journal ArticleDOI
01 Jul 1977-Cell
TL;DR: Results suggest that small numbers of VSV G protein are obligately incorporated into VSV(MuLV) pseudotypes, and there appears to be a stringent requirement for recognition of the viral core by homologous envelope components as the nucleating step in the budding process.

Journal ArticleDOI
TL;DR: Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells.
Abstract: Ecotropic and xenotropic murine leukemia viruses (MuLV's) constitute separate interference groups; within each group there is cross-interference, but between the groups there is no detectable interference. Interference is manifest against pseudotypes in which the vesicular stomatitis virus genome is contained within the coat of one of the murine leukemia viruses. The pseudotypes display the cell specificity of the leukemia viruses: pseudotypes with an ecotropic MuLV coat infect mouse cells but not rabbit or mink cells; pseudotypes with a xenotropic MuLV coat infect rabbit or mink cells well but mouse cells very poorly. Efficient pseudotype formation also occurs between the two MuLV classes, and both the interference patterns and the cell specificity of these pseudotypes are entirely determined by their envelope. Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay. Also, xenotropic MuLV infection could be established in a mouse cell, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells. The major barriers against both xenotropic and ecotropic MuLV therefore are cell surface barriers. Xenogeneic cells probably lack receptors for ecotropic MuLV, but murine cells may either lack receptors for xenotropic MuLV or have receptors that are blocked by endogenous expression of the glycoprotein of endogenous xenotropic MuLV.

Journal ArticleDOI
08 Sep 1977-Nature
TL;DR: DNA synthesised in vitro by purified virions of murine leukaemia virus is infectious and Transfection with reverse transcriptase product shows a single-hit dose response and results in the production of complete, infectious virus.
Abstract: DNA synthesised in vitro by purified virions of murine leukaemia virus is infectious. Neither RNA nor protein is required for infectivity. Transfection with reverse transcriptase product shows a single-hit dose response and results in the production of complete, infectious virus.

Journal ArticleDOI
21 Jul 1977-Nature
TL;DR: To determine the nucleotide sequence relationship between the polio virus RNAs containing and lacking the protein, the 5′-terminus of polio-virus polyribosomal RNA is isolated and sequenced and it is shown here that itsucleotide sequence is identical to that of the 5″-terminal end of virion RNA.
Abstract: POLIO virus is a positive strand virus; it has a mRNA of the same 5′ to 3′ polarity and size as its virion RNA (ref. 1). It also has a protein covalently linked to its 5′-terminal end forming a 5′-terminus of protein–pU–U–A–A–A–A–C–A–Gp (refs 2, 3). This contrasts with other viral RNAs that either have di- or triphosphate 5′-termini (negative strand RNAs)4–6 or m7G5′ppp5′N(m)p… termini (most positive strand RNAs)7,8. The 5′-linked protein on poliovirion RNA is also found on at least two-thirds and perhaps all of the nascent chains of the replicative intermediate isolated from infected cells3. This suggests that the protein is linked to the RNA at a very early stage of replication, perhaps as a primer during the initiation of RNA synthesis2,3. The 5′-terminus of polio virus polyribosomal RNA lacks the protein and instead terminates in pUp… (refs 9, 10). Thus, it is possible that the protein initially present on the newly synthesised RNA chains is cleaved from that fraction of the RNA molecules destined to become mRNA. To determine the nucleotide sequence relationship between the polio virus RNAs containing and lacking the protein, we have isolated and sequenced the 5′-terminus of polio-virus polyribosomal RNA. We show here that its nucleotide sequence is identical to that of the 5′-terminus of virion RNA.

Journal ArticleDOI
TL;DR: Conditions have been developed for reverse transcription by detergent-disrupted virions of Moloney murine leukemia virus which permit synthesis of molecules that appear to be complete transcripts of the 35S RNA subunits.
Abstract: Conditions have been developed for reverse transcription by detergent-disrupted virions of Moloney murine leukemia virus which permit synthesis of molecules that appear to be complete transcripts of the 35S RNA subunits. At limiting Mg2+ concentration, DNA is synthesized in good yield, up to a maximum size of about 2.4 x 10^6 daltons. DNA larger than 2 x 10^6 daltons, taken from alkaline sucrose gradients, has no detectable self-complementarity and was protected from digestion by S1 nuclease to an extent of 90% by annealing to 70S RNA. All size classes ofDNA made in these reactions are primed with RNA, because all are initiated with a pApdA junction. To produce such long molecules, it is necessary to keep the concentration of Mg2+ in the reaction mixture below the total concentration of deoxyribonucleoside triphosphates. Under these conditions, degradation of the RNA template is minimized. The rate of DNA synthesis is also slowed by 30 to 50%, but products longer than 5,000 nucleotides, which are not found otherwise, are completed between 3 and 6 h of reaction.

Journal ArticleDOI
01 May 1977-Cell
TL;DR: The presence of a 5' terminal pppAp structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed, and supports the concept that Uukuniemi virus is a negative strand virus.


Journal ArticleDOI
TL;DR: An apparent precursor to the poliovirion that cosediments with the virion at 150S was identified by its content of VP-0, which has properties previously associated with the provirion, a structure that sedimented at 125S, and it may be an alternate form of provirions.
Abstract: An apparent precursor to the poliovirion that cosediments with the virion at 150S was identified by its content of VP-0. It has properties previously associated with the provirion, a structure that sedimented at 125S, and it may be an alternate form of provirion. Like virions, the 150S precursor binds to and elutes from cells, after which it sediments at about 125S.

Journal ArticleDOI
TL;DR: Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliov virus RNA from each other and from HeLa cell 28S ribosomal RNA.
Abstract: Intracellular poliovirus-specific RNA species can be measured directly by electrophoresis of total cytoplasmic nucleic acids through 1% agarose gels, resulting in the separation of single- and double-stranded forms of poliovirus RNA from each other and from HeLa cell 28S ribosomal RNA. Single-stranded RNA molecules differing by only 15% in length are resolved in this gel system. RNA species can be visualized as fluorescen bands appearing after staining of the gels with ethidium bromide and observation under ultraviolet illumination. The total amount of RNA can be determined by densitometric quantitation of the fluorescent response. In this way, the amount of poliovirus-specific RNA within the cytoplasm of HeLa cells infected for various times has been estimated. At 170-min postinfection, there are 0.67 X 10(5) molecules of single-stranded poliovirus RNA per cell and at 230 min, the amount has increased to 3.7 X 10(5) molecules/cell. Poliovirus double-strnaded RNA reaches a maximum of 0.7 X 10(5) molecules/cell at 330 min after infection.

Journal ArticleDOI
TL;DR: The most suggestive observation that could be interpreted as evidence for terminal transferase in pre-B cells is the occurrence of activity in Abelson virus-induced tumor cells.
Abstract: Terminal transferase remains the only good candidate for a somatic-mutation-producing DNA polymerase. It is clearly found that in immature, but not in mature, T cells. It is also found in a population of bone marrow cells that lack an inducible T-cell marker and that could be B-cell precursors (but might well be unrelated to B cells). The most suggestive observation that could be interpreted as evidence for terminal transferase in pre-B cells is the occurrence of activity in Abelson virus-induced tumor cells. The status of Abelson tumor cells is, however, uncertain.