RESUMO
Previously, we analyzed the effects of point mutations in the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT) and found that some mutations affected both titer and cleavage specificity. We used HIV-1 vectors containing two PPTs and the D116N integrase active-site mutation in a cell-based assay to measure differences in the relative rates of PPT processing and utilization. The relative rates were measured by determining which of the two PPTs in the vector is used to synthesize viral DNA. The results indicate that mutations that have subtle effects on titer and cleavage specificity can have dramatic effects on rates of PPT generation and utilization.
Assuntos
DNA Viral/biossíntese , HIV-1/genética , Mutação Puntual , RNA Viral/genética , Replicação Viral/genética , HIV-1/fisiologia , Humanos , RNA Viral/metabolismo , Replicação Viral/fisiologiaRESUMO
Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.
Assuntos
Transformação Celular Viral , Fibroblastos/metabolismo , Tropomiosina/metabolismo , Animais , Vírus do Sarcoma Aviário , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Conformação Proteica , Tropomiosina/isolamento & purificaçãoRESUMO
Three types of c-ski cDNAs have been isolated from two different chicken cDNA libraries. Sequence comparisons suggest that the cDNAs derive from alternatively spliced mRNAs. A short stretch of sequence homology that exists between c-ski and avian leukosis virus may have played a role in viral transduction.
Assuntos
Galinhas/genética , DNA/genética , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/isolamento & purificação , Éxons , Dados de Sequência Molecular , Oncogenes , Splicing de RNA , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Transdução GenéticaRESUMO
We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.
Assuntos
DNA/análise , Músculos/análise , Tropomiosina/genética , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , DNA/isolamento & purificação , Fibroblastos/análise , Dados de Sequência Molecular , Peso Molecular , Splicing de RNARESUMO
We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/genética , Retroviridae/genética , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Anticorpos , Western Blotting , Células Cultivadas , Embrião de Galinha , Galinhas , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Imunofluorescência , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/análiseRESUMO
We have generated transgenic mouse lines that carry the promoter region of the chicken skeletal muscle alpha (alpha sk) actin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. In adult mice, the pattern of transgene expression resembled that of the endogenous alpha sk actin gene. In most of the transgenic lines, high levels of CAT activity were detected in striated muscle (skeletal and cardiac) but not in the other tissues tested. In striated muscle, transcription of the transgene was initiated at the normal transcriptional start site of the chicken alpha sk actin gene. The region from nucleotides -191 to +27 of the chicken alpha sk actin gene was sufficient to direct the expression of CAT in striated muscle of transgenic mice. These observations suggest that the mechanism of tissue-specific actin gene expression is well conserved in higher vertebrate species.
Assuntos
Actinas/genética , Regiões Promotoras Genéticas , Animais , Galinhas , Cloranfenicol O-Acetiltransferase/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Músculos/metabolismo , Distribuição Tecidual , Transcrição GênicaRESUMO
The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiologia , Músculo Esquelético/fisiologia , Cadeias Leves de Miosina/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA4 , Coração/embriologia , Metilação , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
DNA and RNA polymerases are enzymes that are primarily responsible for copying genetic material in all living systems. The four polymerases whose structures have been determined by X-ray crystallographic methods have significant similarities at the polymerase active site that are indicative of common requirements for polynucleotide synthesis. Structural studies of complexes of the Klenow fragment of Escherichia coli DNA polymerase I, HIV type 1 reverse transcriptase, and rat DNA polymerase beta with DNA are leading to generalized models for catalysis.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Conformação Proteica , Animais , DNA Polimerase I/química , DNA Polimerase I/metabolismo , Humanos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , RatosRESUMO
BACKGROUND: HIV-1 reverse transcriptase (RT) is a major target for anti-HIV drugs. A considerable amount of information about the structure of RT is available, both unliganded and in complex with template-primer or non-nucleoside RT inhibitors (NNRTIs). But significant conformational differences in the p66 polymerase domain among the unliganded structures have complicated the interpretation of these data, leading to different proposals for the mechanisms of polymerization and inhibition. RESULTS: We report the structure of an unliganded RT at 2.7 A resolution, crystallized in space group C2 with a crystal packing similar to that of the RT-NNRTI complexes. The p66 thumb subdomain is folded into the DNA-binding cleft. Comparison of the unliganded RT structures with the DNA-bound RT and the NNRTI-bound RT structures reveals that the p66 thumb subdomain can exhibit two different upright conformations. In the DNA-bound RT, the p66 thumb subdomain adopts an upright position that can be described as resulting from a rigid-body rotation of the p66 thumb along the "thumb's knuckle' located near residues Trp239 (in strand beta 14) and Val317 (in beta 15) compared with the thumb position in the unliganded RT structure. NNRTI binding induces an additional hinge movement of the p66 thumb near the thumb's knuckle, causing the p66 thumb to adopt a configuration that is even more extended than in the DNA-bound RT structure. CONCLUSIONS: The p66 thumb subdomain is extremely flexible. NNRTI binding induces both short-range and long-range structural distortions in several domains of RT, which are expected to alter the position and conformation of the template-primer. These changes may account for the inhibition of polymerization and the alteration of the cleavage specificity of RNase H by NNRTI binding.
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Sítios de Ligação , Biopolímeros , Dados de Sequência Molecular , Conformação Proteica , Difração de Raios XRESUMO
P21waf1/cip1 is a potent inhibitor of cell cycle progression and can inhibit the growth of both normal cells and cells transformed by a number of oncogenes. However, the ability of p21waf1/cip1 to inhibit the growth of cells that overexpress the transcriptional transactivator E2F1 is controversial: it has been reported both that E2F1 can and cannot overcome the block in the cell cycle induced by p21waf1/cip1. To avoid the complications that arise when such experiments are done with permanent cell lines, we tested the effects of overexpressing p21waf/cip1 and E2F1 in primary chicken embryo fibroblasts. In this system very high levels of E2F1 overexpression cause considerable apoptosis; however, the surviving cells still overexpress E2F1. These cells are transformed and their growth is blocked by overexpression of p21waf1/cip1.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Divisão Celular/genética , Ciclinas/genética , Proteínas de Ligação a DNA , Fatores de Transcrição/genética , Animais , Apoptose/genética , Linhagem Celular Transformada , Embrião de Galinha , Inibidor de Quinase Dependente de Ciclina p21 , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G1/genética , Vetores Genéticos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Retroviridae/genéticaRESUMO
To gain a more complete understanding of c-myc regulation in chickens, we have completed the structural characterization of the chicken c-myc gene and have begun to investigate c-myc transcription and protein expression. A comparison of c-myc structure and expression between mammals and birds presents an enigma: there are striking similarities in the pattern of gene expression in the absence of obvious sequence similarities in the controlling elements. We have begun to investigate c-myc and v-myc function using retroviral vectors that differ solely in the Myc proteins that they express. We show that while the overexpression of the smaller c-Myc protein is sufficient to induce morphological transformation in chicken embryo fibroblasts, overexpression of v-Myc provides a stronger signal for cells to enter the cell cycle and is a more potent inducer of apoptosis than c-Myc.
Assuntos
Apoptose/genética , Divisão Celular/genética , Galinhas/genética , Genes myc , Proteína Oncogênica p55(v-myc)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular/genética , Transformação Celular Neoplásica , Embrião de Galinha , Fibroblastos/fisiologia , Fibroblastos/virologia , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/genética , Dados de Sequência Molecular , Proteína Oncogênica p55(v-myc)/biossíntese , Biossíntese de Proteínas , Retroviridae/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
We have used replication-competent retroviral vectors to express avian and murine ras genes in cultured chick embryo fibroblasts (CEF) and in chickens. Since the viral vectors are identical, it is possible to compare the oncogenic potential of the ras genes directly. The normal (12 gly) form of chicken c-Ha-ras is not oncogenic in vivo, nor does high-level expression transform CEF. Expression of murine v-ras or modified forms of chicken c-Ha-ras with either lysine or glutamine at position 12 transforms CEF and causes tumors in birds. However, the oncogenic potential of the transforming ras genes is different; the viruses that express the genes with lysine and glutamine at position 12 cause a distinct spectrum of tumors.
Assuntos
Neoplasias Ósseas/genética , Transformação Celular Viral/genética , Genes ras/genética , Neoplasias Pulmonares/genética , Doenças Musculares/genética , Mutação/genética , Neoplasias Experimentais/genética , Retroviridae/genética , Animais , Neoplasias Ósseas/microbiologia , Galinhas , Neoplasias Pulmonares/microbiologia , Doenças Musculares/microbiologia , Neoplasias Experimentais/microbiologia , TransfecçãoRESUMO
cDNA clones of the ski-related gene, sno, were isolated from a chicken cDNA library and sequenced. In contrast to the human system, from which two forms of sno cDNAs have been isolated, we obtained only one type of chicken sno cDNA, that encoding snoN. The coding region for chicken snoN was inserted into the retroviral vectors RCAS(A) and RCASBP(A) and introduced into chicken embryo fibroblasts (CEFs) or quail embryo cells (QECs). Like the various forms of ski, snoN appears to be localized in the nucleus, and high levels of snoN expression cause transformation of CEFs and muscle differentiation of QECs. In contrast to ski however, low-level expression of snoN cannot induce transformation, and is only weakly myogenic.
Assuntos
Galinhas/genética , DNA/química , Proteínas Oncogênicas de Retroviridae/genética , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Embrião de Galinha , Vetores Genéticos , Dados de Sequência Molecular , Codorniz , Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/análiseRESUMO
In normal cells, cell growth and division is controlled by the interplay between proto-oncogenes and tumor suppressor genes. Cancer cells usually have both activated an oncogene and have lost a functional tumor suppressor gene. High level expression of a tumor suppressor, p53, can block the growth of cancer cells. waf1/cip1 is transactivated by the tumor suppressor p53 and the p21waf1/cip1 protein is itself a suppressor of cell growth. To test the effect of growth suppression genes on the growth of cells transformed by individual oncogenes, we have used replication-competent retroviral vectors to induce high level expression of p53 and p21waf1/cip1. Overexpression of p21waf1/cip1 arrests the growth of chicken embryo fibroblasts (CEF) transformed by v-Src, tf-Ras, c-Mos and c-Myc. These data suggest that p21waf1/cip1 might be a useful tool in gene therapy for human cancer.
Assuntos
Transformação Celular Neoplásica/patologia , Ciclinas/fisiologia , Inibidores Enzimáticos , Oncogenes , Animais , Divisão Celular , Embrião de Galinha , Inibidor de Quinase Dependente de Ciclina p21 , Fase G1 , Humanos , Coelhos , Proteína Supressora de Tumor p53/análiseRESUMO
Four novel sulfonic acid polymers were evaluated for their in vitro HIV-1 and HIV-2 reverse transcriptase (RT) inhibitory activity and found to be equipotent against both RTs. The aromatic polymers demonstrated IC50 values that were approximately 10(3)-fold lower than those observed with the aliphatic polymers. Among the aromatic polymers, poly(4-styrenesulfonic acid) (PSS) (MW 8000; IC50 = 0.02 microgram/ml) was 3-fold more potent than poly(anetholesulfonic acid) (PAS) of approximately the same molecular weight range. The activity of PSS polymers increased in proportion to the size of the polymers and, relative to suramin, activity could be enhanced over 200-fold. These polymers also inhibited the cytopathic effect of HIV-1 at concentrations that were non-toxic to MT-4 cells. The potent RT inhibitory properties of these stable sulfonic acid polymers suggest that structure-activity studies are warranted to yield agents capable of inhibiting multiple stages of the viral process.
Assuntos
Antivirais/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , DNA Polimerase Dirigida por RNA , Inibidores da Transcriptase Reversa , Ácidos Sulfônicos/farmacologia , Animais , Linhagem Celular , Transcriptase Reversa do HIV , Polímeros/farmacologia , Relação Estrutura-Atividade , Suramina/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
We have determined the organization of the chicken tropomyosin 1 gene by sequencing the cloned genomic DNA. The single-copy gene spans approximately 11,000 bases and includes 12 exons. Comparison of cDNA and genomic sequences demonstrates that three tissue-specific tropomyosins are encoded by the gene: a 284 amino acid skeletal muscle beta-tropomyosin, a 284 amino acid smooth muscle tropomyosin, and a 248 amino acid non-muscle (fibroblast) beta-tropomyosin. Skeletal and smooth muscle transcripts use the same putative promoter and transcription initiation site. However, they are alternatively spliced to generate mRNAs that differ in the region giving rise to amino acids 188 to 213 and 258 through the poly(A) site. The fibroblast transcript uses a promoter, initiation site and first exon that is distinct from that used for both the smooth and the skeletal muscle transcripts. However, beyond the first exon the fibroblast transcript undergoes splicing and polyadenylation that is identical with the smooth muscle transcript.
Assuntos
Genes , Variação Genética , Músculo Liso/metabolismo , Músculos/metabolismo , Tropomiosina/genética , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , DNA/genética , Sondas de DNA , Éxons , Fibroblastos/metabolismo , Biblioteca Gênica , Dados de Sequência Molecular , Peso Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227-427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT). We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244-322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.
Assuntos
Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-2/enzimologia , Ribonuclease H/metabolismo , Dimerização , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-2/genética , Modelos Moleculares , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/química , Ribonuclease H/genética , Moldes GenéticosRESUMO
Based on structural analyses and on the behavior of mutants, we suggest that the polymerase domain of HIV-1 reverse transcriptase (RT) plays a critical role in holding and appropriately positioning the template-primer both at the polymerase active site and at the RNase H active site. For RT to successfully copy the viral RNA genome, RNase H must cleave the RNA with absolute precision. We believe that a combination of the structure of the template-primer and its precise positioning are responsible for the specific cleavages RNase H makes. We have proposed that resistance of HIV-1 RT to nucleoside analogs involves a subtle repositioning of the template-primer. This hypothesis is based on both structural and biochemical analyses. Mutations that confer resistance to nucleoside analogs do not cluster at the polymerase active site; however, they are in positions where they could alter the interaction between RT and the template-primer. If, as we have hypothesized, the polymerase domain is primarily responsible for positioning the template-primer and RNase H cleavage depends on this positioning, it should be possible to use RNase H cleavage to monitor at least some of the major changes in the position of the template-primer. We have used three assays (polymerase, RNase H, and strand transfer) to investigate the effects of mutations in the polymerase domain, including mutations that confer resistance to nucleotide analogs, on HIV-1 RT. All three assays involve RNA sequences derived from the viral genome. The data show that alterations in the polymerase domain, in particular, mutations that are in positions that would be expected to alter the interaction of RT with the template-primer, can alter both the efficiency and specificity of RNase H cleavage. These results are discussed in light of the structure of HIV-1 RT.
Assuntos
Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Mutagênese , RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Sítios de Ligação , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Mutations that confer resistance to nucleoside analogs do not cluster around the deoxynucleotide triphosphate (dNTP) binding site. Instead, these mutations appear to lie along the groove in the enzyme where the template-primer binds. Based on such structural data and on complementary biochemical analyses, it has been suggested that resistance to nucleoside analogs involves repositioning of the template-primer. We have prepared mutations in HIV-2 RT that are the homologs of mutations that confer resistance to nucleoside analogs in HIV-1 RT. Analysis of the behavior of HIV-2 RT mutants (Leu74Val, Glu89Gly, Ser215Tyr, Leu74Val/Ser215Tyr and Glu89Gly/Ser215Tyr) in vitro confirms the results obtained with HIV-1 RT: resistance is a function of the length of the template overhang. These analyses also suggest that the homolog in HIV-2 RT of one of the mutations that confers resistance to AZT in HIV-1 RT (Thr215Tyr) confers resistance by repositioning of the template-primer.
Assuntos
DNA Viral/química , Desoxirribonucleotídeos/metabolismo , HIV-1/enzimologia , HIV-2/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Primers do DNA/química , Nucleotídeos de Desoxiguanina/metabolismo , Didesoxinucleotídeos , Resistência Microbiana a Medicamentos/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Modelos Moleculares , Mutação , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes , Moldes Genéticos , Nucleotídeos de Timina/metabolismoRESUMO
Retroviral revXerse transcriptases (RTs) have an associated RNase H activity that can cleave RNA-DNA duplexes with considerable precision. We believe that the structure of the RNA-DNA duplexes in the context of RT determines the specificity of RNase H cleavage. To test this idea, we treated three related groups of synthetic RNA-DNA hybrids with either Moloney murine leukemia virus (MLV) RT or human immunodeficiency virus type 1 (HIV-1) RT. All of the hybrids were prepared using the same 81-base RNA template. The first series of RNase H substrates was prepared with complementary DNA oligonucleotides of different lengths, ranging from 6 to 20 nucleotides, all of which shared a common 5' end and were successively shorter at their 3' ends. The second series of oligonucleotides had a common 3' end but shorter 5' ends. The DNA oligonucleotides in the third series were all 20 bases long but had non-complementary stretches at either the 5' end, 3' end, or both ends. Several themes have emerged from the experiments with these RNA-DNA duplexes. (1) Both HIV-1 RT and MLV RT cleave fairly efficiently if the duplex region is at least eight bases long, but not if it is shorter. (2) Although, under the conditions we have used, both enzymes require the substrate to have a region of RNA-DNA duplex, both MLV RT and HIV-1 RT can cleave RNA outside the region that is part of the RNA-DNA duplex. (3) The polymerase domain of HIV-1 RT uses certain mismatched segments of RNA-DNA to position the enzyme for RNase H cleavage, whereas the polymerase domain of MLV RT does not use the same mismatched segments to define the position for RNase H cleavage. (4) For HIV-1 RT, a mismatched region near the RNase H domain can interfere with RNase H cleavage; cleavage is usually (but not always) more efficient if the mismatched segment is deleted. These results are discussed in regard to the structure of HIV-1 RT and the differences between HIV-1 RT and MLV RT.