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1.
Gene Ther ; 13(19): 1382-90, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16724096

RESUMO

The ability to control (trans)gene expression is important both for basic biological research and applications such as gene therapy. In vivo use of the inducible tetracycline (Tc)-regulated gene expression system (Tet-On system) is limited by its low sensitivity for the effector doxycycline (dox). We used viral evolution to optimize this Escherichia coli-derived regulatory system for its function in mammalian cells. The components of the Tet-On system (the transcriptional activator rtTA and its tetO DNA binding site) were incorporated into the human immunodeficiency virus (HIV)-1 genome to control viral replication. Prolonged culturing of this HIV-rtTA virus resulted in virus variants that acquired mutations in the rtTA gene. Some of these mutations enhance the transcriptional activity and dox-sensitivity of the rtTA protein. This improvement was observed with different tetO-containing promoters and was independent of the episomal or chromosomal status of the target gene. Combination of these beneficial mutations resulted in greatly improved rtTA variants that are seven-fold more active and 100-fold more dox-sensitive than the original Tet-On system. Furthermore, some of the new Tet-On systems are responsive to Tc and minocycline. Importantly, these rtTA variants show no activity in the absence of dox. The optimized rtTA variants are particularly useful for in vivo applications that require a more sensitive or more active Tet-On system.


Assuntos
Antibacterianos/uso terapêutico , Evolução Biológica , Regulação Viral da Expressão Gênica/genética , Terapia Genética/métodos , HIV-1/genética , Tetraciclina/uso terapêutico , Western Blotting/métodos , Linhagem Celular , Clonagem Molecular , Terapia Combinada , DNA/análise , Doxiciclina/uso terapêutico , Escherichia coli , Expressão Gênica , Engenharia Genética , Células HeLa , Humanos , Mutação , Proteínas/genética , Linfócitos T/metabolismo , Transativadores/genética , Transgenes , Replicação Viral/genética
2.
J Virol ; 74(5): 2227-38, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10666253

RESUMO

Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA(3)(Lys) molecule that binds with its 3'-terminal 18 nucleotides to the fully complementary primer-binding site (PBS) on the viral RNA genome. Besides this complementarity, annealing of the primer may be stimulated by additional base-pairing interactions between other parts of the tRNA molecule and viral sequences flanking the PBS. According to the RNA secondary structure model of the HIV-1 leader region, part of the PBS sequence is involved in base pairing to form a small stem-loop structure, termed the U5-PBS hairpin. This hairpin may be involved in the process of reverse transcription. To study the role of the U5-PBS hairpin in the viral replication cycle, we introduced mutations in the U5 region that affect the stability of this structured RNA motif. Stabilization and destabilization of the hairpin significantly inhibited virus replication. Upon prolonged culturing of the virus mutant with the stabilized hairpin, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the U5-PBS hairpin. The thermodynamic stability of the U5-PBS hairpin apparently has to stay within narrow limits for efficient HIV-1 replication. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and assembly of the virions with a normal RNA content is not affected by the mutations within the U5-PBS hairpin. We show that stabilization of the hairpin reduced the amount of tRNA primer that is annealed to the PBS. Destabilization of the hairpin did not affect tRNA annealing, but the viral RNA-tRNA complex was less stable. These results suggest that the U5-PBS hairpin is involved in correct placement of the tRNA primer on the viral genome. The analysis of virus mutants and revertants and the RNA structure probing experiments presented in this study are consistent with the existence of the U5-PBS hairpin as predicted in the RNA secondary structure model.


Assuntos
Genoma Viral , HIV-1/genética , RNA de Transferência de Lisina/genética , RNA Viral/química , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Primers do DNA , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA de Transferência de Lisina/metabolismo , RNA Viral/genética , Relação Estrutura-Atividade , Transfecção , Replicação Viral
3.
Virology ; 263(1): 55-69, 1999 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-10544082

RESUMO

HIV-1 LAI is a syncytium-inducing (SI) virus with a broad host cell range. We previously isolated a LAI variant that improved replication in the SupT1 T cell line due to mutations within the C1 and C4 constant regions of the Env protein. We now report that this variant exhibits a severely restricted host cell range, as replication in other T cell lines and primary cells was abolished. Several Env-mediated functions were analyzed to provide a mechanistic explanation for this selective adaptation. The change in host cell tropism was not caused by a switch to a SupT1-specific coreceptor. Biosynthesis of the variant Env glycoprotein was not improved in SupT1 cells, and in fact a small defect in intracellular Env processing was observed. SupT1 infection assays did not reveal an improved Env function either, and a dramatic loss of infectivity was measured with other cell types. The Env-mutated HIV-1 reached an approximately fivefold higher level of virus production in SupT1 cells at the peak of infection. Unlike the LAI virus, the variant did not trigger the formation of syncytia. Our combined results suggest that the HIV-1 variant allows the infected host cell to survive longer, thus producing more viral progeny. The intricate virus-cell interaction results in a balance between optimal virus replication and host cell survival, causing a cytopathic SI isolate to evolve toward a nonsyncytium-inducing (NSI) phenotype in cell culture. These findings may help explain the absence of SI variants in the initial phase of HIV-1 infection, and the results dispute the notion that HIV-1 evolution should always go from the NSI to SI phenotype.


Assuntos
Células Gigantes/fisiologia , HIV-1/fisiologia , Linfócitos T/virologia , Proteínas do Envelope Viral/genética , Replicação Viral , Adaptação Fisiológica , Linhagem Celular , Efeito Citopatogênico Viral , HIV-1/genética , Humanos , Mutação , Proteínas do Envelope Viral/metabolismo , Cultura de Vírus
4.
J Virol ; 73(1): 81-91, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9847310

RESUMO

Some retroviruses with an extended repeat (R) region encode the polyadenylation signal within the R region such that this signal is present at both the 5' and 3' ends of the viral transcript. This necessitates differential regulation to either repress recognition of the 5' polyadenylation signal or enhance usage of the 3' signal. The human immunodeficiency virus type 1 (HIV-1) genome encodes an inherently efficient polyadenylation signal within the 97-nucleotide R region. Polyadenylation at the 5' HIV-1 polyadenylation site is inhibited by downstream splicing signals, and usage of the 3' polyadenylation site is triggered by an upstream enhancer element. In this paper, we demonstrate that this on-off switch of the HIV-1 polyadenylation signal is controlled by a secondary RNA structure that occludes part of the AAUAAA hexamer motif, which we have termed the polyA hairpin. Opening the 5' hairpin by mutation triggered premature polyadenylation and caused reduced synthesis of viral RNA, indicating that the RNA structure plays a pivotal role in repression of the 5' polyadenylation site. Apparently, the same hairpin structure does not interfere with efficient usage of the 3' polyadenylation site, which may be due to the presence of the upstream enhancer element. However, when the 3' hairpin was further stabilized by mutation, we measured a complete loss of 3' polyadenylation. Thus, the thermodynamic stability of the polyA hairpin is delicately balanced to allow nearly complete repression of the 5' site yet efficient activation of the 3' site. This is the first report of regulated polyadenylation that is mediated by RNA secondary structure. A similar hairpin motif that occludes the polyadenylation signal can be proposed for other lentiviruses and members of the spumaretroviruses, suggesting that this represents a more general gene expression strategy of complex retroviruses.


Assuntos
HIV-1/genética , Poli A/química , RNA Mensageiro/química , RNA Viral/química , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
J Virol ; 72(11): 9217-23, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9765469

RESUMO

The human immunodeficiency virus type 1 RNA genome contains a terminal repeat (R) sequence that encodes the TAR hairpin motif, which has been implicated in Tat-mediated activation of transcription. More recently, a variety of other functions have been proposed for this structured RNA element. To determine the replicative roles of the 5' and 3' TAR hairpins, we analyzed multiple steps in the life cycle of wild-type and mutant viruses. A structure-destabilizing mutation was introduced in either the 5', the 3', or both TAR motifs of the proviral genome. As expected, opening of the 5' TAR hairpin caused a transcription defect. Because the level of protein expression was not similarly reduced, the translation of this mRNA was improved. No effect of the 3' hairpin on transcription and translation was measured. Mutations of the 5' and 3' hairpin structures reduced the efficiency of RNA packaging to similar extents, and RNA packaging was further reduced in the 5' and 3' TAR double mutant. Upon infection of cells with these virions, a reduced amount of reverse transcription products was synthesized by the TAR mutant. However, no net reverse transcription defect was observed after correction for the reduced level of virion RNA. This result was confirmed in in vitro reverse transcription assays. These data indicate that the 5' and 3' TAR motifs play important roles in several steps of the replication cycle, but these structures have no significant effect on the mechanism of reverse transcription.


Assuntos
Repetição Terminal Longa de HIV , HIV-1/crescimento & desenvolvimento , HIV-1/genética , Sequência de Bases , Linhagem Celular , Estabilidade de Medicamentos , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Transcrição Gênica , Transfecção
6.
Virology ; 244(2): 552-62, 1998 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-9601523

RESUMO

The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple regulatory elements that fold into stable hairpin structures. Because extensive secondary structure can block the scanning of ribosomes, an alternative mechanism for HIV translation seems feasible. To study the mechanism of HIV-1 mRNA translation, a start codon was introduced in the leader region that will usurp scanning ribosomes. This upstream AUG mutation (uAUG) inhibited HIV gene expression, indicating that HIV-1 mRNA translation occurs via the regular scanning mechanism. Revertant viruses with increased replication capacity were obtained upon prolonged culturing of the mutant virus. To our surprise, the introduced start codon had not been inactivated in these phenotypic revertants. Instead, these revertants contain additional mutations in the envelope (Env) protein that stimulated HIV-1 replication. These second-site Env mutations did not specifically overcome the gene expression defect of the uAUG mutant, as the replication capacity of other HIV-1 mutants with an unrelated defect could also be improved. The uAUG construct appears to be a unique tool in forced HIV-1 adaptation studies because the deleterious uAUG mutation is stably maintained in the progeny, yielding phenotypic revertants with second-site mutations elsewhere in the viral genome.


Assuntos
Genes env , HIV-1/genética , HIV-1/fisiologia , Mutação , Sequência de Bases , Linhagem Celular , Códon de Iniciação/genética , Expressão Gênica , Genoma Viral , Humanos , Biossíntese de Proteínas , RNA Viral/genética , Replicação Viral/genética
7.
AIDS Res Hum Retroviruses ; 13(13): 1101-7, 1997 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9282815

RESUMO

Several polyanionic reagents such as dextran sulfates, heparin sulfates, and negatively charged proteins have been reported to exhibit anti-HIV activity in vitro. Particularly potent inhibition has been reported for the milk protein beta-lactoglobulin (betaLG) on modification by 3-hydroxyphthalic anhydride (3HP). The introduction of multiple negatively charged carboxyl groups along the polypeptide backbone obviously leads to repulsion within the protein molecule and this is likely to affect the specific tertiary, and perhaps also secondary, structure of the protein. We used several biophysical techniques to probe the structural changes that occur on 3HP modification of betaLG. The results suggest that the protein becomes largely unstructured on chemical modification. Although a profound anti-HIV activity was measured for 3HP-betaLG, similar antiviral effects were observed with two other 3HP-modified milk proteins, alpha-lactalbumin and alpha(S2)-casein, but not with the unmodified proteins. Most potent inhibition of HIV-1 replication was obtained with 3HP-modified alpha-lactalbumin, which also demonstrated the least cytotoxicity. These combined results indicate that HIV inhibition is a general property of negatively charged polypeptides and do not support a model in which the negatively charged 3HP-betaLG protein interacts in a structure-specific manner with the CD4 cell surface receptor for HIV-1 entry.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Proteínas do Leite/farmacologia , Animais , Ânions/farmacologia , Fármacos Anti-HIV/química , Varredura Diferencial de Calorimetria , Caseínas/química , Caseínas/farmacologia , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Lactalbumina/química , Lactalbumina/farmacologia , Lactoglobulinas/química , Lactoglobulinas/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/virologia , Proteínas do Leite/química , Anidridos Ftálicos/química , Conformação Proteica , Espalhamento de Radiação
8.
J Gen Virol ; 78 ( Pt 4): 837-40, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9129656

RESUMO

Retroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3' end of this tRNA and a sequence near the 5' end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18 nucleotides of the tRNA primer are copied into the viral cDNA, thereby regenerating the PBS sequence of the progeny. Thus, the PBS sequence reveals which primer was used. Human immunodeficiency viruses are known to replicate efficiently with tRNA(Lys3) as primer. Examination of the PBS sequence in natural and laboratory isolates indicates that a variant tRNA(Lys) is occasionally used as primer. This variant, for which the murine genomic sequence was described previously, was termed tRNA(Lys5) and differs from tRNA(Lys3) at five nucleotide positions. These results suggest that HIV uses both tRNA(Lys3) and tRNA(Lys5) molecules as primer, causing a switch of the PBS sequence.


Assuntos
HIV-1/genética , RNA de Transferência de Lisina/genética , Transcrição Gênica , Replicação Viral/genética , Sequência de Bases , Sítios de Ligação/genética , Infecções por HIV/virologia , Humanos , Dados de Sequência Molecular , Análise de Sequência
9.
J Virol ; 71(3): 2346-56, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9032371

RESUMO

The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple hairpin motifs. The repeat region of the leader, which is reiterated at the 3' end of the RNA molecule, encodes the well-known TAR hairpin and a second hairpin structure with the polyadenylation signal AAUAAA in the single-stranded loop [the poly(A) hairpin]. The fact that this poly(A) stem-loop structure and its thermodynamic stability are well conserved among HIV and simian immunodeficiency virus isolates, despite considerable divergence in sequence, suggests a biological function for this RNA motif in viral replication. Consistent with this idea, we demonstrate that mutations that alter the stability of the stem region or delete the upper part of the hairpin do severely inhibit replication of HIV type 1. Whereas destabilizing mutations in either the left- or right-hand side of the base-paired stem interfere with virus replication, the double mutant, which allows the formation of new base pairs, replicates more rapidly than the two individual virus mutants. Upon prolonged culturing of viruses with an altered hairpin stability, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the poly(A) hairpin. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and reverse transcription of the genomic RNAs are not affected by mutation of the 5' poly(A) hairpin. We show that the genomic RNA content of the virions is reduced by destabilization of this poly(A) hairpin but not by stabilization or truncation of this structure. These results suggest that the formation of the poly(A) hairpin structure at the 5' end of the genomic RNA molecule is necessary for packaging of viral genomes into virions and/or stability of the virion RNA.


Assuntos
HIV-1/genética , Poli A , RNA Viral , Replicação Viral , Sequência de Bases , Linhagem Celular , Sequência Conservada , Expressão Gênica , Genes Virais , Genoma Viral , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Mutagênese , Transcrição Gênica , Células Tumorais Cultivadas
10.
Nucleic Acids Res ; 25(5): 940-7, 1997 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-9023102

RESUMO

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.


Assuntos
Evolução Molecular Direcionada , HIV-1/genética , RNA Viral , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Linhagem Celular , Genoma Viral , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Poli A , Temperatura , Replicação Viral
11.
J Mol Biol ; 252(1): 59-69, 1995 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-7666433

RESUMO

Retroviruses convert their RNA genome into a DNA form by means of reverse transcription. According to the current model of reverse transcription, two strand transfer reactions are needed to synthesize a full-length DNA genome. Because reverse transcription is initiated close to the 5' end of the RNA genome, the first strand transfer translocates the minus-strand cDNA to the 3' end of the viral genome. This jump is facilitated by the presence of a perfect repeat element (R) at both ends of a retroviral genome. Strand transfer has been extensively studied in in vitro systems with purified reverse transcriptase enzyme (RT) and nucleic acid donor and acceptor templates. In this study, we set out to test several parameters of the strand transfer reaction as it occurs in cells infected with the human immunodeficiency virus (HIV-1). We constructed mutant HIV-1 genomes with 3' R acceptor sequences that were specifically altered either in length or structure. Analysis of the replication characteristics of the mutant viruses indicates that repeats much shorter than the wild-type 97-nucleotides R region can efficiently act as acceptors during reverse transcription. Furthermore, the introduction of excessively stable hairpin structures within the 3' R element did only marginally affect the strand transfer efficiency. We also analysed the DNA forms inherited upon infection of cells with HIV-1 templates with multiple 3' R copies. These experiments indicate that various 3' R repeats can serve as acceptor during minus-strand DNA transfer.


Assuntos
DNA Viral/biossíntese , DNA Viral/metabolismo , HIV-1/genética , Replicação Viral , Sequência de Bases , Primers do DNA/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Viral/genética , Transcrição Gênica , Vírion
12.
J Virol ; 69(5): 3090-7, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7707537

RESUMO

Replication of the human immunodeficiency virus type 1 (HIV-1) and other retroviruses involves reverse transcription of the viral RNA genome into a double-stranded DNA. This reaction is primed by the cellular tRNA(3Lys) molecule, which binds to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). In order to study the specificity of primer usage, we constructed a set of HIV-1 mutants with altered PBS sites corresponding to other tRNA species (tRNA(Ile), tRNA(1,2Lys), tRNA(Phe), tRNA(Pro), tRNA(Trp)). These mutant viruses were able to replicate, although with delayed replication kinetics compared with wild-type HIV-1. Identification of the tRNA species associated with the genomic RNA demonstrated binding of tRNAs complementary to the new PBS sites. However, the occupancy of the mutant PBS sites by these new primers was reduced and correlated well with the replication potential of the mutant viruses. These results suggest that the PBS sequence is not sufficient for annealing of the tRNA primer. Upon prolonged culturing, all mutants reverted to the wild-type PBS(3Lys) sequence. Minor sequence changes in the nucleotides flanking the PBS site indicate that these reversions resulted from annealing of the wild-type tRNA(3Lys) primer onto the mutant PBS sites, followed by copying of part of the tRNA(3Lys) sequence during reverse transcription. Furthermore, the reversion efficiency of the different PBS mutants was found to correlate with their tRNA(Lys)3 binding capacity. A remarkable reversion pathway was observed for the PBSPro variant (PBSPro-->PBSIle-->PBSwt). This pathway can be explained by efficient base pairing of tRNA(Ile) to PBSPro, followed by annealing of tRNA(3Lys) onto the PBSIle intermediate. These results demonstrate that HIV-1 is dedicated to the tRNA(3Lys) primer and that factors other than the PBS sequence determine the selective primer usage of this retrovirus.


Assuntos
HIV-1/genética , HIV-1/fisiologia , Mutação , Replicação Viral/genética , Replicação Viral/fisiologia , Sequência de Bases , Sítios de Ligação/genética , Primers do DNA/genética , Primers do DNA/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo , RNA Viral/genética , Transcrição Gênica , Transfecção
13.
J Gen Virol ; 76 ( Pt 4): 845-53, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9049330

RESUMO

The TAR domain is an RNA secondary structure element within the leader transcript of the human immunodeficiency virus type 1 (HIV-1) virus. TAR RNA forms the binding site for the viral trans-activator protein Tat and cellular co-factors that are involved in induction of the LTR transcriptional promoter. Here, we report that mutations in the single-stranded bulge- and loop-domains of TAR RNA impair the ability of the virus to replicate in T cell lines. Revertant viruses were isolated upon prolonged culturing and analysed through sequencing. The reversion data confirm the importance of both bulge and loop as sequence-specific recognition motifs. We also analysed the replication phenotype of a mutant HIV-1 virus with a substitution in the -19/-3 promoter region. This mutant displayed delayed infection kinetics compared to the wild-type virus, and revertants with increased replication potential could be isolated. Interestingly, all revertants had acquired an additional mutation at position -2. Primer extension analyses revealed that an upstream shift in transcription start site usage was induced by the -19/-3 substitution. This effect was compensated for by the nucleotide substitution near the RNA start site.


Assuntos
Repetição Terminal Longa de HIV , HIV-1/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Células COS , Linhagem Celular , Células Cultivadas , DNA Viral , HIV-1/fisiologia , Células HeLa , Humanos , Leucócitos Mononucleares/citologia , Dados de Sequência Molecular , Mutagênese , Linfócitos T/citologia , Linfócitos T/virologia , Replicação Viral
14.
Virology ; 207(1): 276-81, 1995 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-7755727

RESUMO

A comparative sequence analysis of part of the RNA genome containing the poly(A) signal of different groups of immunodeficiency viruses, including human types 1 and 2, simian types mandrill, african green monkey, and sykes, reveals the conservation of certain structural features despite the divergence in sequence. In all cases, the AAUAAA signal was found to be flanked by nucleotide segments that can basepair, thus forming a hairpin structure with the poly(A) signal in the single-stranded loop. The fact that both this stem-loop structure and its thermodynamic stability are well conserved suggests a biological function for this structure motif [corrected]. Consistent with this idea, we demonstrate that stabilization or destabilization of the stem region does severely inhibit the replication potential of the HIV-1 virus.


Assuntos
HIV-1/química , HIV-2/química , Conformação de Ácido Nucleico , Poli A/química , RNA Viral/química , Vírus da Imunodeficiência Símia/química , Algoritmos , Animais , Composição de Bases , Sequência de Bases , Linhagem Celular , Variação Genética/genética , HIV-1/genética , HIV-1/fisiologia , HIV-2/genética , Humanos , Dados de Sequência Molecular , Mutação/fisiologia , Filogenia , Primatas , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Vírus da Imunodeficiência Símia/genética , Replicação Viral
15.
EMBO J ; 13(11): 2650-9, 1994 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-8013464

RESUMO

Human immunodeficiency virus type 1 (HIV-1) RNA contains an extended hairpin structure at the 5' end (the TAR element) that is essential for viral replication. The upper part of the stem-loop structure binds the virally encoded transcriptional activator protein Tat and cellular co-factors, but no clear function for the lower stem region has been established. Here, we report that mutant HIV-1 viruses with base substitutions in the lower stem region are dead, most likely at the level of transcription from an integrated provirus. By using large amounts of these mutant DNA constructs for transfections, revertant viruses with a great variety of genetic changes (point mutations, short deletions) could be isolated in prolonged culture experiments that lasted over 6 months. The pattern and evolution of these changes supported the notion that base-pairing of the lower stem region is essential for optimal HIV-1 replication. The functional and genetic plasticities of this RNA domain and the HIV-1 long terminal repeat promoter are discussed.


Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/genética , Conformação de Ácido Nucleico , Mutação Puntual/genética , RNA Viral/química , Composição de Bases , Sequência de Bases , Evolução Biológica , Linhagem Celular , Análise Mutacional de DNA , DNA Viral/genética , HIV-1/crescimento & desenvolvimento , HIV-1/fisiologia , Humanos , Cinética , Dados de Sequência Molecular , Provírus , RNA Viral/análise , Transcrição Gênica , Replicação Viral/genética
16.
J Virol ; 68(6): 3830-40, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8189520

RESUMO

The architecture of a retroviral genome presents some unusual features for transcriptional regulation because of duplication of the transcriptional control sequences in the 5' and 3' long terminal repeats (LTRs). We have studied the transcriptional activity of the 5' and 3' LTRs of human immunodeficiency virus type 1 (HIV-1) vectors. Using full-length HIV molecular clones, we demonstrate that both LTRs function as Tat-inducible promoters. However, the absolute levels of transcription were found to be much higher for the 5' LTR than for the 3' LTR promoter. When transcription was assayed for an integrated HIV-1 provirus, we also found that the upstream 5' LTR element was the major transcriptional promoter. 3' LTR transcription, however, can be triggered by inactivation of the 5' LTR promoter. Likewise, 5' LTR transcription is induced in constructs lacking a functional 3' LTR promoter. This phenomenon of promoter suppression may have important implications for the design of HIV-based retrovirus vectors.


Assuntos
Repetição Terminal Longa de HIV , HIV-1/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Produtos do Gene tat/genética , Vetores Genéticos , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese , Provírus/genética , RNA Viral/genética , Supressão Genética , Linfócitos T/microbiologia , Transcrição Gênica , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana
17.
Nucleic Acids Res ; 22(2): 137-44, 1994 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-7510065

RESUMO

Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides.


Assuntos
DNA Viral/biossíntese , HIV-1/genética , RNA Viral/genética , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Gênica/fisiologia , Sequência de Bases , Linhagem Celular , Repetição Terminal Longa de HIV , Transcriptase Reversa do HIV , Dados de Sequência Molecular , Mutação , Recombinação Genética , Moldes Genéticos
18.
Nucleic Acids Res ; 21(22): 5020-4, 1993 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-8255755

RESUMO

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.


Assuntos
Evolução Biológica , Produtos do Gene tat/metabolismo , Genoma Viral , HIV-1/genética , Mutagênese , RNA Viral/genética , Sequência de Bases , DNA Viral , Repetição Terminal Longa de HIV , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Viral/química , Sequências Reguladoras de Ácido Nucleico , Produtos do Gene tat do Vírus da Imunodeficiência Humana
19.
Virology ; 188(1): 391-5, 1992 May.
Artigo em Inglês | MEDLINE | ID: mdl-1566581

RESUMO

HIV-1 NEF genes were isolated directly from peripheral blood lymphocyte DNA of two HIV-1-infected individuals and cloned into an HXB-2-infectious molecular clone. The effect of NEF on virus production in T-cell lines and primary human lymphocytes was studied. Naturally occurring NEF accelerates virus production in primary human lymphocytes, but not in T-cell lines.


Assuntos
Genes nef , HIV-1/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Viral , Produtos do Gene nef/química , Produtos do Gene nef/genética , Repetição Terminal Longa de HIV , HIV-1/fisiologia , Humanos , Cinética , Linfócitos/microbiologia , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de Sequência , Linfócitos T/microbiologia , Replicação Viral/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana
20.
J Virol ; 66(2): 757-65, 1992 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1731110

RESUMO

Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non-syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1.


Assuntos
Células Gigantes/fisiologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-1/fisiologia , Replicação Viral , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Células Cultivadas , Quimera , Clonagem Molecular , Variação Genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Linfócitos T , Transfecção
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