RESUMEN

The persistence of HIV-infected cells in individuals on suppressive combination antiretroviral therapy (cART) presents a major barrier for curing HIV infections. HIV integrates its DNA into many sites in the host genome; we identified 2410 integration sites in peripheral blood lymphocytes of five infected individuals on cART. About 40% of the integrations were in clonally expanded cells. Approximately 50% of the infected cells in one patient were from a single clone, and some clones persisted for many years. There were multiple independent integrations in several genes, including MKL2 and BACH2; many of these integrations were in clonally expanded cells. Our findings show that HIV integration sites can play a critical role in expansion and persistence of HIV-infected cells.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Infecciones por VIH/virología , VIH/fisiología , Factores de Transcripción/genética , Integración Viral/genética , Latencia del Virus/genética , Antirretrovirales/uso terapéutico , Células Clonales/virología , ADN Viral/análisis , ADN Viral/genética , ADN Viral/metabolismo , Genoma Humano , VIH/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Humanos , ARN Viral/análisis , ARN Viral/genética , ARN Viral/metabolismo

RESUMEN

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.

Asunto(s)

ADN Viral/biosíntesis , VIH-1/genética , Mutación Puntual , ARN Viral/genética , Replicación Viral/genética , VIH-1/fisiología , Humanos , ARN Viral/metabolismo , Replicación Viral/fisiología

RESUMEN

The synthesis of retroviral DNA is initiated near the 5' end of the RNA. DNA synthesis is transferred from the 5' end to the 3' end of viral RNA in an RNase H-dependent step. In the case of human immunodeficiency virus type 1 (HIV-1) (and certain other retroviruses that have complex secondary structures at the ends of the viral RNA), there is the possibility that DNA synthesis can lead to a self-priming event that would block viral replication. The extent of RNase H cleavage must be sufficient to allow the strand transfer reaction to occur, but not so extensive that self-priming occurs. We have used a series of model RNA substrates, with and without a 5' cap, to investigate the rules governing RNase H cleavage at the 5' end of the HIV-1 genome. These in vitro RNase H cleavage reactions produce an RNA fragment of the size needed to block self-priming but still allow strand transfer. The cleavages seen in vitro can be understood in light of the structure of HIV-1 reverse transcriptase in a complex with an RNA/DNA substrate.

Asunto(s)

Genoma Viral , VIH-1/genética , Ribonucleasa H/metabolismo , Secuencia de Bases , ADN Viral/genética , Hidrólisis , Regiones Promotoras Genéticas , ARN Viral/genética

RESUMEN

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.

Asunto(s)

Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-2/enzimología , Ribonucleasa H/metabolismo , Dimerización , Transcriptasa Inversa del VIH/genética , VIH-1/genética , VIH-2/genética , Modelos Moleculares , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa H/química , Ribonucleasa H/genética , Moldes Genéticos

RESUMEN

Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used--photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N(2)-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5' extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the "open" configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.

Asunto(s)

Transcriptasa Inversa del VIH/genética , VIH-1/genética , Reactivos de Enlaces Cruzados , ADN/química , ADN/genética , Transcriptasa Inversa del VIH/química , VIH-1/química , Humanos , Conformación de Ácido Nucleico , Especificidad por Sustrato , Moldes Genéticos

RESUMEN

After minus-strand strong-stop DNA (-sssDNA) is synthesized, the RNA template is degraded by the RNase H activity of the reverse transcriptase (RT), generating a single-stranded DNA. The 3' end of -sssDNA from HIV-1 can form a hairpin; this hairpin will self-prime in vitro. We previously used a model substrate, -R ssDNA, which corresponds to the 3' end of the -sssDNA of HIV-1, to show that the self-priming of this model substrate could be prevented by annealing a 17-nt-long DNA oligonucleotide to the 3' end of -R ssDNA in the presence of HIV-1 nucleocapsid (NC) protein. Similar model substrates were prepared for HIV-2 and HTLV-1; the R regions of these two viruses are longer and form more complex structures than the R region of the HIV-1 genome. However, the size of the R region and the complexity of the secondary structures they can form do not affect self-priming or its prevention. The efficiency of the self-priming is related to the relative stabilities of the conformations of -R ssDNA that can and cannot induce self-priming. For the three viruses (HIV-1, HIV-2, and HTLV-1), the size of the DNA oligonucleotide needed to block self-priming in the presence of NC is similar to the expected size of the piece of RNA left after degradation of the RNA template during reverse transcription. We also found that when the 3' end of -R ssDNA is annealed to a complementary DNA oligonucleotide, it is a good substrate for efficient nonspecific strand transfer to other single-stranded DNA molecules.

Asunto(s)

ADN de Cadena Simple/química , ADN Viral/química , VIH-1/genética , VIH-2/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleocápside/genética , Oligodesoxirribonucleótidos/química , Cloruro de Potasio , ARN Viral/química , Retroviridae/genética

RESUMEN

When human immunodeficiency virus type 1 (HIV-1) is selected for resistance to 3TC, the methionine normally present at position 184 is replaced by valine or isoleucine. Position 184 is the X of the conserved YXDD motif; positions 185 and 186 form part of the triad of aspartic acids at the polymerase active site. Structural and biochemical analysis of 3TC-resistant HIV-1 reverse transcriptase (RT) led to a model in which a beta-branched amino acid at position 184 would act as a steric gate. Normal deoxynucleoside triphosphates (dNTPs) could still be incorporated; the oxathiolane ring of 3TCTP would clash with the beta branch of the amino acid at position 184. This model can also explain 3TC resistance in feline immunodeficiency virus and human hepatitis B virus. However, it has been reported (14) that murine leukemia viruses (MLVs) with valine (the amino acid present in the wild type), isoleucine, alanine, serine, or methionine at the X position of the YXDD motif are all resistant to 3TC. We prepared purified wild-type MLV RT and mutant MLV RTs with methionine, isoleucine, and alanine at the X position. The behavior of these RTs was compared to those of wild-type HIV-1 RT and of HIV-1 RT with alanine at the X position. If alanine is present at the X position, both MLV RT and HIV-1 RT are relatively resistant to 3TCTP in vitro. However, the mutant enzymes were impaired relative to their wild-type counterparts; there appears to be steric hindrance for both 3TCTP and normal dNTPs.

Asunto(s)

Fármacos Anti-VIH/farmacología , Citidina Trifosfato/farmacología , VIH-1/efectos de los fármacos , Lamivudine/farmacología , Virus de la Leucemia Murina de Moloney/efectos de los fármacos , ADN Polimerasa Dirigida por ARN/genética , Alanina , Animales , Citidina Trifosfato/análogos & derivados , Didesoxinucleótidos , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Isoleucina , Lamivudine/análogos & derivados , Metionina , Conformación Molecular , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , Mutación Puntual , ADN Polimerasa Dirigida por ARN/química , Replicación Viral/efectos de los fármacos

RESUMEN

The amount of excess polymerase and RNase H activity in human immunodeficiency virus type 1 virions was measured by using vectors that undergo a single round of replication. Vectors containing wild-type reverse transcriptase (RT), vectors encoding the D110E mutation to inactivate polymerase, and vectors encoding mutations D443A and E478Q to inactivate RNase H were constructed. 293 cells were cotransfected with different proportions of plasmids encoding these vectors to generate phenotypically mixed virions. The resulting viruses were used to infect human osteosarcoma cells, and the relative infectivity of the viruses was determined by measuring transduction of the murine cell surface marker CD24, which is encoded by the vectors. The results indicated that there is an excess of both polymerase and RNase H activities in virions. Viral replication was reduced to 42% of wild-type levels in virions with where half of the RT molecules were predicted to be catalytically active but dropped to 3% of wild-type levels when 25% of the RT molecules were active. However, reducing RNase H activity had a lesser effect on viral replication. As expected, based on previous work with murine leukemia virus, there was relatively inefficient virus replication when the RNase H and polymerase activities were encoded on separate vectors (D110E plus E478Q and D110E plus D443A). To determine how virus replication failed when polymerase and RNase H activities were reduced, reverse transcription intermediates were measured in vector-infected cells by using quantitative real-time PCR. The results indicated that using the D11OE mutation to reduce the amount of active polymerase reduced the number of reverse transcripts that were initiated and also reduced the amounts of products from the late stages of reverse transcription. If the E478Q mutation was used to reduce RNase H activity, the number of reverse transcripts that were initiated was reduced; there was also a strong effect on minus-strand transfer.

Asunto(s)

Transcriptasa Inversa del VIH/metabolismo , VIH-1/fisiología , ADN Polimerasa Dirigida por ARN/metabolismo , Ribonucleasa H/metabolismo , Células Cultivadas , Vectores Genéticos , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/patogenicidad , Humanos , Datos de Secuencia Molecular , Mutación , ADN Polimerasa Dirigida por ARN/genética , Ribonucleasa H/genética , Transfección , Replicación Viral

Asunto(s)

Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Inhibidores de la Transcriptasa Inversa/farmacología , Dimerización , VIH-1/enzimología , Humanos , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Relación Estructura-Actividad

RESUMEN

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 x 10(6) CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

Asunto(s)

Vectores Genéticos , Retroviridae/genética , Transfección , Animales , Virus del Sarcoma Aviar/genética , Línea Celular , Células Cultivadas , Embrión de Pollo , Virus de la Leucemia Murina/genética , Ratones , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/genética , Replicación Viral

RESUMEN

Inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) are widely used in the treatment of HIV infection. Loviride (an alpha-APA derivative) and HBY 097 (a quinoxaline derivative) are two potent non-nucleoside RT inhibitors (NNRTIs) that have been used in human clinical trials. A major problem for existing anti-retroviral therapy is the emergence of drug-resistant mutants with reduced susceptibility to the inhibitors. Amino acid residue 103 in the p66 subunit of HIV-1 RT is located near a putative entrance to a hydrophobic pocket that binds NNRTIs. Substitution of asparagine for lysine at position 103 of HIV-1 RT is associated with the development of resistance to NNRTIs; this mutation contributes to clinical failure of treatments employing NNRTIs. We have determined the structures of the unliganded form of the Lys103Asn mutant HIV-1 RT and in complexes with loviride and HBY 097. The structures of wild-type and Lys103Asn mutant HIV-1 RT in complexes with NNRTIs are quite similar overall as well as in the vicinity of the bound NNRTIs. Comparison of unliganded wild-type and Lys103Asn mutant HIV-1 RT structures reveals a network of hydrogen bonds in the Lys103Asn mutant that is not present in the wild-type enzyme. Hydrogen bonds in the unliganded Lys103Asn mutant but not in wild-type HIV-1 RT are observed between (1) the side-chains of Asn103 and Tyr188 and (2) well-ordered water molecules in the pocket and nearby pocket residues. The structural differences between unliganded wild-type and Lys103Asn mutant HIV-1 RT may correspond to stabilization of the closed-pocket form of the enzyme, which could interfere with the ability of inhibitors to bind to the enzyme. These results are consistent with kinetic data indicating that NNRTIs bind more slowly to Lys103Asn mutant than to wild-type HIV-1 RT. This novel drug-resistance mechanism explains the broad cross-resistance of Lys103Asn mutant HIV-1 RT to different classes of NNRTIs. Design of NNRTIs that make favorable interactions with the Asn103 side-chain should be relatively effective against the Lys103Asn drug-resistant mutant.

Asunto(s)

Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Mutación Missense/genética , Inhibidores de la Transcriptasa Inversa/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/farmacología , Acetofenonas/química , Acetofenonas/metabolismo , Acetofenonas/farmacología , Sustitución de Aminoácidos/genética , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Estabilidad de Enzimas , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína , Quinoxalinas , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Termodinámica

RESUMEN

Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5'-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

Asunto(s)

Fármacos Anti-VIH/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Inhibidores de la Transcriptasa Inversa/metabolismo , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Fármacos Anti-VIH/farmacología , Didesoxinucleótidos , Resistencia a Medicamentos , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , Humanos , Modelos Moleculares , Mutagénesis , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Timina/farmacología , Zidovudina/farmacología

RESUMEN

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Asunto(s)

Transcriptasa Inversa del VIH/química , Oligodesoxirribonucleótidos/química , Oligorribonucleótidos/química , Purinas/química , Cristalografía , Cartilla de ADN/química , VIH-1/crecimiento & desarrollo , Modelos Moleculares , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Poli A/química , Poli T/química , Poli dA-dT/química , Estructura Cuaternaria de Proteína , Ribonucleasa H/química , Especificidad por Sustrato , Propiedades de Superficie , Sincrotrones , Transcripción Genética , Replicación Viral

RESUMEN

Escherichia coli RNase H has a basic extension that is involved in binding nucleic acid substrates. This basic extension is present in the RNase H of Moloney murine leukemia virus reverse transcriptase (MLV RT), but has been deleted from the RNase H of HIV-1 RT. Previous work showed that removing the basic loop from MLV RT (the mutant is called DeltaC) blocked viral replication; however, DeltaC MLV RT retained RNase H activity in an in situ gel assay. We prepared recombinant DeltaC MLV RT and compared its activity to wild-type MLV RT. The DeltaC mutant is impaired in both polymerase and RNase H activity; the pattern of defects suggests that the basic loop is involved in the binding of MLV RT to a heteropolymeric template-primer.

Asunto(s)

Dominio Catalítico , ADN Polimerasa Dirigida por ADN/genética , Virus de la Leucemia Murina de Moloney/enzimología , ADN Polimerasa Dirigida por ARN/genética , Ribonucleasa H/genética , Animales , ADN Polimerasa Dirigida por ADN/metabolismo , Ratones , Mutación , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasa H/metabolismo

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA(3)(Lys) primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5' end of the RNA that remained annealed to the 3' end of the minus-strand strong-stop DNA only if NC was present in the reaction.

Asunto(s)

ADN Viral/biosíntesis , Genoma Viral , VIH-1/genética , Ácidos Nucleicos Heterodúplex , ARN Viral/metabolismo , Secuencia de Bases , ADN de Cadena Simple/biosíntesis , Escherichia coli/enzimología , Duplicado del Terminal Largo de VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/metabolismo , Heparina/farmacología , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/metabolismo , ARN Viral/química , Ribonucleasa H/metabolismo , Moldes Genéticos

RESUMEN

If foreign genes are ubiquitously expressed in mice using a viral vector, expression is abrogated by CD8(+) cells in 2 to 4 weeks. However, if the expression of the genes is confined to skeletal muscle cells, the CD8(+) T-cell response is much weaker and expression is maintained for more than 6 weeks. These data show that restricting the expression of foreign genes to skeletal muscle cells and presumably to other cells that are inefficient at antigen presentation can prolong the expression of a foreign gene product.

Asunto(s)

Fosfatasa Alcalina/biosíntesis , Virus de la Leucosis Aviar/genética , Expresión Génica , Vectores Genéticos , Músculo Esquelético/metabolismo , Músculo Esquelético/virología , Proteínas Recombinantes/biosíntesis , Fosfatasa Alcalina/genética , Animales , Linfocitos T CD8-positivos/inmunología , Ratones , Músculo Esquelético/citología , Ovalbúmina/química , Ovalbúmina/inmunología , Biosíntesis de Péptidos , Péptidos/genética , Péptidos/inmunología

RESUMEN

Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (-sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with -sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic -sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3' end of the -sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.

Asunto(s)

ADN Viral/genética , VIH-1/fisiología , Proteínas de la Nucleocápside/genética , ARN Viral/genética , Replicación Viral , Transcriptasa Inversa del VIH/genética , Humanos , Oligonucleótidos/genética

RESUMEN

Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.

Asunto(s)

Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Lamivudine/metabolismo , Lamivudine/farmacología , Sustitución de Aminoácidos/genética , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Sitios de Unión , Catálisis , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxicitosina/farmacología , Desoxirribonucleótidos/metabolismo , Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Modelos Moleculares , Mutación/genética , Ensayos de Protección de Nucleasas , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , ARN/biosíntesis , ARN/química , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/metabolismo , Sales (Química)/farmacología , Electricidad Estática , Moldes Genéticos , Transcripción Genética

RESUMEN

We compared the fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and two RT mutants, Y115F and Y115V. Although neither mutation had a large effect on the overall fidelity of the enzyme, both mutations altered the spectrum of mutations and the precise nature of the mutational hot spots. The effects of Y115V were greater than those of Y115F. When we compared the behavior of the wild-type enzyme with published data, we found that, in contrast to what has been published, misalignment/slippage could account for only a small fraction of the mutations we observed. We also found that a preponderance of the mutations (both transitions and transversions) resulted in the insertion of an A. Because we were measuring DNA-dependent DNA synthesis (plus-strand synthesis), this bias could contribute to the A-rich nature of the HIV-1 genome.

Asunto(s)

Sustitución de Aminoácidos , Transcriptasa Inversa del VIH/química , Secuencia de Aminoácidos , Disparidad de Par Base , Secuencia de Bases , ADN Bacteriano/química , Escherichia coli/química , Transcriptasa Inversa del VIH/genética , Datos de Secuencia Molecular , Mutación Missense , beta-Galactosidasa/genética

RESUMEN

We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116-Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.

Asunto(s)

Desoxirribonucleótidos/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Sitios de Unión , Desoxirribonucleótidos/genética , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Cinética , Inhibidores de la Transcriptasa Inversa/farmacología