RESUMO
Retrovirus particles contain a small, basic protein, the nucleocapsid (NC) protein, that possesses 'nucleic acid chaperone' activity--that is, the NC protein can catalyze the rearrangement of a nucleic acid molecule into the conformation that has the maximal number of base pairs. The molecular mechanism that underlies this effect is not understood. Because the chaperone activity is apparently crucial during the infectious process, NC is a potential target for antiviral therapy.
Assuntos
Ácidos Nucleicos/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Retroviridae/genética , Replicação Viral , Sequência de Aminoácidos , Produtos do Gene gag/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Conformação Proteica , RNA Viral/genética , RNA Viral/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Produtos do Gene pol/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , DNA Nucleotidiltransferases/química , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Produtos do Gene pol/química , Immunoblotting , Integrases , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Ligação Proteica , CoelhosRESUMO
Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.
Assuntos
DNA Nucleotidiltransferases/genética , Escherichia coli/genética , HIV-1/enzimologia , Proteínas Recombinantes de Fusão/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Western Blotting , Clonagem Molecular , DNA Nucleotidiltransferases/imunologia , DNA Nucleotidiltransferases/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Anticorpos Anti-HIV/análise , HIV-1/imunologia , Humanos , Integrases , Plasmídeos , Testes de Precipitina , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , SolubilidadeAssuntos
Proteínas de Bactérias/biossíntese , RNA de Transferência/análise , Acilação , Sítios de Ligação , Isótopos de Carbono , Escherichia coli , Código Genético , Magnésio , Biossíntese Peptídica , Fenilalanina/metabolismo , Polinucleotídeos/análise , Ligação Proteica , Puromicina/análise , RNA Bacteriano/análise , Ribossomos/análise , Ribossomos/metabolismo , Fatores de Tempo , TrítioRESUMO
Virions produced by cells in the presence of actinomycin D (Act D virions) contain reverse transcriptase but are deficient in 70S genomic RNA. To assess the role of genomic RNA in encapsidation of a functional reverse transcriptase and to study the interaction of the enzyme and its template in the cores of intact virions, the reverse transcriptase enzymes of normal and Act D virions were compared. The enzymes were indistinguishable by column chromatography, sedimentation velocity, or template/primer preferences. In addition, these enzymes showed equal sensitivity to inactivation by antibodies directed against Rauscher murine leukemia virus DNA polymerase. The enzymes from Act D and normal virions had similar thermal decay rates and were both protected against heat denaturation by natural and synthetic template/primers. By these criteria, the DNA polymerase molecules synthesized and assembled into virions in the absence of genomic RNA are identical to those packaged under normal conditions. Additional studies designed to measure protection of reverse transcriptase by genomic RNA were carried out by comparing the thermal lability of the enzyme in intact Act D and normal virions. The thermal decay rate of reverse transcriptase in Act D virions was identical to that in control virions. In contrast to the lability of the virion-associated enzyme, however, genomic RNA in control virions was stable to heat treatment.
Assuntos
Vírus AKR da Leucemia Murina/enzimologia , Vírus da Leucemia Murina/enzimologia , RNA Viral/fisiologia , DNA Polimerase Dirigida por RNA/metabolismo , Vírus AKR da Leucemia Murina/crescimento & desenvolvimento , Centrifugação com Gradiente de Concentração , Cromatografia , Cromatografia por Troca Iônica , Dactinomicina/farmacologia , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , RNA Viral/análise , DNA Polimerase Dirigida por RNA/análiseRESUMO
Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.
Assuntos
Arbovírus/análise , Eletroforese Descontínua , RNA Viral/análise , Vírus da Floresta de Semliki/análise , Adenosina , Animais , Centrifugação com Gradiente de Concentração , Embrião de Galinha , Galinhas , Técnicas de Cultura , Fibroblastos , Peso Molecular , Ácidos Fosfóricos , Isótopos de Fósforo , RNA Viral/isolamento & purificação , Vírus da Floresta de Semliki/crescimento & desenvolvimento , Sindbis virus/análise , Sindbis virus/crescimento & desenvolvimento , Sacarose , Trítio , Uridina , Cultura de VírusRESUMO
Mammalian type C retroviruses use translational suppression to synthesize the enzymes which function in virus replication. The UAG termination codon at the end of the coding region for the viral core proteins is translated as glutamine at a frequency of approximately 5%, allowing synthesis of the enzymes as part of a large fusion protein. This unusual mechanism has several benefits for the virus: first, it modulates the relative levels of synthesis of the core proteins and the enzymes. This is essential for the proper assembly of the virus particle, since the fusion protein alone is apparently unable to assemble into particles. Second, the presence of the core protein moiety in the fusion protein probably provides a mechanism for targeting the enzymes to the virus particle. The mechanism of the suppression phenomenon is now under investigation. Recent studies have revealed that suppression in the viral context is dependent upon a complex cis-acting signal in the viral mRNA, including a pseudoknot beginning 9 nucleotides 3' of the termination codon. In addition, studies with viral mutants have shown that UAA and UGA, like UAG, are efficiently suppressed in the presence of this signal, and have identified the amino acids used in the suppression of these termination codons in reticulocyte lysates. In several cases, this analysis revealed the existence of previously unknown suppressor tRNAs. One important question which has not been answered is whether the suppression mechanism used by the virus has a parallel in the synthesis of host proteins.
Assuntos
Retroviridae/genética , Supressão Genética , Sequência de Bases , Códon/genética , Proteínas de Fusão gag-pol/biossíntese , Proteínas de Fusão gag-pol/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Viral/genética , Retroviridae/fisiologia , Replicação Viral/genéticaRESUMO
At the 3' end of all retroviral genomes there is a short, highly conserved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determinants for in vitro priming by the human immunodeficiency virus type 1 (HIV-1) PPT. We show that when the PPT is removed and placed into different nucleotide contexts, new priming sites are produced at the precise 3' end of the PPT. In addition, we find that a hybrid consisting of a 15- or 20-nucleotide RNA primer annealed to a 35-nucleotide DNA template is competent for initiation of plus-strand synthesis with HIV-1 reverse transcriptase. Thus, no cis-acting elements appear to be required for priming activity. Changes at the 5' end of the PPT have no effect on primer function, whereas the identity of bases at the 3' end is crucial. A primer containing only the 6 G residues from the 3' end of the wild-type PPT sequence and 9 bases of random sequence at the 5' end functions like a wild-type PPT. A short hybrid having a similar helical structure but a primary sequence different from that of the PPT is cleaved imprecisely, resulting in initiation of synthesis at multiple sites; however, total primer extension is close to the wild-type level. We conclude that helical structure as well as the presence of particular bases at the 3' end of the PPT is essential for PPT function.
Assuntos
DNA Viral/genética , HIV-1/genética , Purinas , Replicação do DNA , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Murine leukemia virus particles assembled in actinomycin D-treated cells were detected by determination of reverse transcriptase [RNA-dependent DNA polymerase (nucleotidyltransferase)] activity and by radioimmunoassay of the major virion protein, p30. The levels of enzyme activity and p30 protein were both 30-40% relative to the control over an 8 hr period, whereas after 3 or 4 hr infectivity was reduced by 95%. Thus, virions produced in the absence of RNA synthesis represent a fairly homogeneous population of defective particles. Although RNA synthesis is not necessary for virus assembly, protein synthesis is required. Treatment of cells with 10 mug/ml of cycloheximide reduced virus production by 80-85% within 2 hr, and by greater than 95% at later times. As might be expected from this finding, viral protein synthesis accompanies virus assembly in actinomycin D-treated cells. Newly synthesized proteins associated with the defective particles were identical with those found in standard virions and were present in the correct proportions. The results demonstrate that viral mRNA persists in cells in which RNA synthesis is blocked and continues to direct viral protein synthesis with a functional half-life of approximately 6-8 hr. Since viral mRNA is not packaged in virions even when viral RNA synthesis is shut off [Levin et al. (1974) J. Virol. 14, 152-161], we propose that murine leukemia virus-infected cells contain two nonequilibrating pools of intracellular viral RNA molecules, one associated with polyribosomes and one which is encapsidated into extracellular particles.
Assuntos
Vírus AKR da Leucemia Murina/metabolismo , Vírus da Leucemia Murina/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Vírus Defeituosos/análise , Vírus Defeituosos/crescimento & desenvolvimento , Cinética , DNA Polimerase Dirigida por RNA/biossíntese , Proteínas Virais/análiseRESUMO
The 4S RNA contained in RNA tumor virus particles consists of a selected population of host tRNA's. However, the mechanism by which virions select host tRNA's has not been elucidated. We have considered a model which specifies that 35S genomic RNA determines which tRNA's are to be encapsidated as well as the relative amounts of these tRNA's within the virion. The model was tested by comparing the free 4S RNA composition of normal murine leukemia virus (MuLV) particles and noninfectious virions from actinomycin D (ActD)-treated cells, which are deficient in genomic RNA (ActD virions). Viral 4S RNA was analyzed by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the patterns obtained for control and ActD 4S RNA were identical to each other and were clearly distinct from the cell 4S RNA pattern. The viral patterns had three prominent areas of radioactivity. One of the spots was identified on the basis of its oligonucleotide fingerprint as tRNA (Pro), the primer for MuLV RNA-directed DNA synthesis. These results were obtained with two different MuLV strains, AKR and Moloney, each grown in SC-1 cells. The demonstration that ActD virions contain primer tRNA and in general exhibit the characteristic MuLV tRNA pattern rather than the complete representation of cell 4S RNA leads to the conclusion that genomic RNA is not the major determinant in selective packaging of host tRNA's. A possible role for one or more viral proteins, including reverse transcriptase, is suggested.
Assuntos
Vírus AKR da Leucemia Murina/metabolismo , Genes Virais , Vírus da Leucemia Murina/metabolismo , Vírus da Leucemia Murina de Moloney/metabolismo , RNA de Transferência/metabolismo , RNA Viral/genética , Linhagem Celular , Dactinomicina/farmacologia , RNA Viral/análise , RNA Viral/fisiologiaRESUMO
The role of reverse transcriptase in selective encapsidation of the murine leukemia virus (MuLV) tRNA primer, tRNAPro, was investigated by examining the tRNA composition of several nonconditional pol mutants. One mutant, clone 23, which contains an altered polymerase about 40% smaller than the wild-type enzyme (B. I. Gerwin et al., J. Virol. 31:741-751, 1979) had a typical viral tRNA pattern, including normal levels of tRNAPro in free and 70S-associated 4S RNA. Another class of mutants, produced by Moloney murine leukemia virus-infected cell clone M13 and subclone M13/1, does not contain any detectable polymerase protein (A. Shields et al., Cell 14:601-609, 1978) and was found to have reduced amounts of tRNAPro in free 4S RNA. However, the level of tRNAPro associated with the genome was normal in the mutant virions. These results suggest that the reverse transcriptase protein is involved in the initial selection of tRNA primer during virus assembly, but not in the subsequent association of this tRNA with genomic RNA.
Assuntos
Vírus da Leucemia Murina/genética , RNA de Transferência/genética , RNA Viral/genética , DNA Polimerase Dirigida por RNA/genética , Vírus da Leucemia Murina/enzimologia , Mutação , Prolina , Ligação Proteica , RNA de Transferência/metabolismo , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Serial passage of Semliki Forest virus (SFV) in chicken embryo cells had little effect on SFV yield; however, high multiplicity infection of murine cells with one of the late passage pools (passage 9 SFV) resulted in a virus yield 10- to 20-fold lower than that obtained with earlier passage virus and 80-fold lower than the corresponding yield in chicken cells. This effect was accompanied by a striking decrease in the levels of 42S and 26S RNA and by increased proportions of a small single-stranded viral RNA (molecular weight, 9 x 10(5)) and of a low-molecular-weight replicative form. There was also a reduction in the number of specific membranous structures previously associated with the group A arbovirus replication complex. These results suggested that passage 9 SFV contained defective interfering particles which were detected more readily after one passage in a murine indicator host cell. Identical results were obtained with two different murine cell lines: one a leukemia virus-free clone of AKR cells and the other JLS-V9 cells chronically infected with Rauscher leukemia virus. Host production of RNA tumor virus particles apparently did not affect arbovirus replication.
Assuntos
Vírus Defeituosos/crescimento & desenvolvimento , Vírus da Floresta de Semliki/crescimento & desenvolvimento , Interferência Viral , Replicação Viral , Adenosina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Dactinomicina/farmacologia , Eletroforese em Gel de Poliacrilamida , Leucemia Experimental , Camundongos , Peso Molecular , Radioisótopos de Fósforo , RNA Viral/análise , RNA Viral/biossíntese , Vírus Rauscher/crescimento & desenvolvimento , Vírus da Floresta de Semliki/análise , Vírus da Floresta de Semliki/metabolismo , Especificidade da Espécie , Trítio , Uridina/metabolismo , Cultura de VírusRESUMO
Molecular hybridization techniques were used to examine the stability of viral message and virion precursor RNA in murine leukemia virus-infected cells treated with actinomycin D. Under the conditions used, viral RNA synthesis was inhibited, but viral protein synthesis continued, and the cells produced noninfectious particles (actinomycin D virions) lacking genomic RNA (J. G. Levin and M. J. Rosenak, Proc. Natl. Acad. Sci. U.S.A. 73:1154-1158, 1976). Analysis of total RNA in virions revealed that the amount of hybridizable viral RNA decreased steadily after the addition of actinomycin D and by 8 h was 10% of the control value. Studies on fractionated viral RNA showed that this low level of hybridization is due to residual 70S RNA in the virion population. The results indicated that viral RNA which is destined to be encapsidated into virions has a half-life of approximately 3 to 4 h. In contrast, other intracellular virus-specific RNA molecules appeared to be quite stable and persisted for a long period of time, with a half-life of at least 12 h. These observations support the idea that two independent functional pools of 35S viral RNA exist within the infected cell: one serving as message and the other as precursor to virion RNA. The existence of two viral RNA pools was further documented by the finding that 12 h after the addition of actinomycin D, when virion precursor RNA was depleted, 35S and 21S viral nRNA species could be identified in polyribosomal RNA as well as in total polyadenylated cell RNA. Surprisingly, 35S and mRNA declined more rapidly than did 21S mRNA, which appeared to be increased in amount.
Assuntos
Vírus AKR da Leucemia Murina/metabolismo , Vírus da Leucemia Murina/metabolismo , Precursores de Ácido Nucleico/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Animais , Linhagem Celular , Dactinomicina/farmacologia , Meia-Vida , Camundongos , Poli A/metabolismo , RNA/metabolismo , RNA Ribossômico/metabolismo , Vírion/metabolismoRESUMO
The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).
Assuntos
Vírus AKR da Leucemia Murina/genética , Endonucleases/genética , Vírus da Leucemia Murina/genética , DNA Polimerase Dirigida por RNA/genética , Proteínas dos Retroviridae/genética , Vírus AKR da Leucemia Murina/imunologia , Endonucleases/imunologia , Escherichia coli/genética , Genes , Genes Virais , Conformação Proteica , DNA Polimerase Dirigida por RNA/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas dos Retroviridae/imunologiaRESUMO
The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activities of AKR murine leukemia virus (MuLV) enzymes. In addition to the wild type, an RNase H-minus RT missing the entire RNase H domain and two other mutants having abnormal polymerase:RNase H ratios were expressed. These mutants include (i) a chimeric protein in which the MuLV RNase H domain was replaced by the entire Escherichia coli RNase H sequence and (ii) an RT with a 126 amino acid deletion in a region analogous to the "connection" subdomain in the p66 subunit of human immunodeficiency virus type 1 RT (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992) Science 256, 1783-1790). With the wild-type RT, the major RNase H cleavage reaction was coordinated with DNA synthesis and occurred at a position corresponding to 15 nucleotides from the 3'-terminus of the DNA primer. Additional cleavages closer to the 5'-end of the RNA were explained in terms of a model relating binding of the RNA.DNA hybrid substrate and enzyme structure. The chimeric RT behaved like E. coli RNase H, exhibited 300-fold higher RNase H activity than wild-type RT, and was limited in its ability to synthesize DNA. Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed. The RNase H domain appeared to function independently of the polymerase domain, supporting the idea that the proper spatial relationship between the two active centers was disrupted by the mutation. Taken together, our results indicate that alteration of the normal polymerase:RNase H ratio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains.
Assuntos
Escherichia coli/enzimologia , Vírus da Leucemia Murina/enzimologia , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Deleção de Sequência , Animais , Sítios de Ligação , Clonagem Molecular , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Cinética , Vírus da Leucemia Murina/genética , Camundongos , Camundongos Endogâmicos AKR , Conformação de Ácido Nucleico , Plasmídeos , Poli A/química , Poli A/metabolismo , Poli T/química , Poli T/metabolismo , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonuclease H/isolamento & purificaçãoRESUMO
Expression of the murine leukemia virus pol gene occurs by translational readthrough of an in-frame UAG codon between the gag and pol coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.
Assuntos
Vírus da Leucemia Murina de Moloney/genética , Terminação Traducional da Cadeia Peptídica , Sequência de Aminoácidos , Arginina , Códon , Cisteína , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Genes Supressores , Glutamina , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA de Transferência/genética , TriptofanoRESUMO
Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm(3)) and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid.oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only (1/5) the level found in controls. This observation correlated with a marked reduction in infectivity. Kinetic studies on the appearance of labeled RNA in banded virions revealed that within the first hour after addition of actinomycin D, particles contained 60 to 70S RNA and two low-molecular-weight RNA species corresponding to 8 and 4S RNA. After approximately 1 h of incubation with actinomycin D, 60 to 70S RNA could not be detected and 4S RNA was the predominant species. These findings suggest that murine leukemia virus particles assembled in the presence of actinomycin D are deficient in 60 to 70S viral RNA.
Assuntos
Dactinomicina/farmacologia , Vírus da Leucemia Murina/crescimento & desenvolvimento , RNA Viral/biossíntese , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , DNA Polimerase Dirigida por RNA/análise , Vírus Rauscher/crescimento & desenvolvimento , Retroviridae/crescimento & desenvolvimento , Trítio , UridinaRESUMO
The pol gene of murine leukemia virus and other mammalian type C retroviruses is expressed by read-through suppression of an in-frame UAG codon which separates the gag and pol coding regions. In this study, we have analyzed the sequence requirements for read-through suppression by placing different portions of wild-type and mutant viral sequences from the gag-pol junction between reporter genes and testing transcripts of these constructs for suppression in reticulocyte lysates. We find that the read-through signal is contained within the first 57 nucleotides on the 3' side of the UAG codon. Our results indicate that the identities of six conserved bases in the eight-nucleotide, purine-rich sequence immediately downstream of the UAG codon are critical for suppression, as is the existence of a pseudoknot structure spanning the next 49 nucleotides. Thus, read-through suppression depends on a complex, bipartite signal in the mRNA.
Assuntos
Genes gag , Vírus da Leucemia Murina de Moloney/genética , RNA Mensageiro/genética , RNA Viral/genética , Supressão Genética , Regiões Terminadoras Genéticas , Animais , Sequência de Bases , Clonagem Molecular , Códon/genética , Proteínas de Fusão gag-pol/genética , Genes pol , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Reticulócitos/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.