Replication of Human Immunodeficiency Viruses Engineered with Heterologous Tat-Transactivation Response Element Interactions

Xie, Baode; Wainberg, Mark A.; Frankel, Alan D.

doi:10.1128/jvi.77.3.1984-1991.2003

Cited by 14 publications

(23 citation statements)

References 58 publications

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“…A second pair of oligonucleotides (tat1, 5Ј-AAA CAC CCC GGG TCC CAG CCG AAA ACC GCG TGC ACC AAC TGC TAC TGC AAA AAA TGC TGC TTC-3Ј; and tat2, 5Ј-GCC GTA AGA GAT TCC TAG GGC TTT GGT GAT GAA GCA AAA CCT GGC AGT GGA AGC AGC ATT TTT TGC-3Ј) was annealed, double-stranded DNA was synthesized, and the product was digested with HinfI (underlined sequence in tat2). The two HinfI-digested fragments were ligated, digested with XmaI and MluI (underlined sequences in tat1 and tat3), and then ligated into an XmaI-MluI-digested HIV-1 proviral vector (pR7BtatHTAR [60]) to generate fusions to amino acid 48 of HIV Tat. The ligation mixture was electroporated into Escherichia coli, ϳ10 5 colonies were collected, and plasmid DNA was prepared with a Qiagen Mix kit.…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type HIV-1 (from the R7/3 proviral plasmid) (17) and viral variants were cultured in 293T human embryonic kidney cells maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin or in MT-4 cells maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS and 1% penicillin-streptomycin, as previously described (60). For chloramphenicol acetyltransferase (CAT) assay experiments, HeLa or NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 1% penicillin-streptomycin.…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, when selective pressure was exerted on viral replication by engineering a noncognate Tat-TAR interaction into HIV-1, mutations readily arose that generated high-affinity interactions (60). Here we have constructed combinatorial peptide libraries within HIV-1 and have used viral replication to select for tight binders to HIV TAR.…”

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confidence: 99%

“…Interestingly, however, the JDV Tat ARM can act as a chameleon that binds RNA in two distinct binding modes, by using a cyclin T1-independent, ␤-hairpin conformation to bind BIV TAR and a cyclin T1-dependent, extended conformation to bind HIV TAR, utilizing one arginine for specific recognition (49). In the context of HIV-1 replication, hybrid viruses engineered with various HIV, BIV, and JDV Tat and TAR combinations have been shown to replicate only when high-affinity binding strategies are possible, including the chameleon behavior of the JDV ARM (60).…”

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confidence: 99%

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Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System

Xie

Calabro

Wainberg

et al. 2004

J Virol

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The interaction between the arginine-rich motif (ARM) of the human immunodeficiency virus (HIV) Tat protein and TAR RNA is essential for Tat activation and viral replication. Two related lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), also require Tat ARM-TAR interactions to mediate activation, but the viruses have evolved different RNA-binding strategies. Interestingly, the JDV ARM can act as a "chameleon," adopting both the HIV and BIV TAR binding modes. To examine how RNA-protein interactions may evolve in a viral context and possibly to identify peptides that recognize HIV TAR in novel ways, we devised a retroviral system based on HIV replication to amplify and select for RNA binders. We constructed a combinatorial peptide library based on the BIV Tat ARM and identified peptides that, like the JDV Tat ARM, also function through HIV TAR, revealing unexpected sequence characteristics of an RNAbinding chameleon. The results suggest that a retroviral screening approach may help identify high-affinity TAR binders and may provide new insights into the evolution of RNA-protein interactions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System

Xie

Calabro

Wainberg

et al. 2004

J Virol

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“…Among the most well-characterized ARM peptide-RNA interactions are the Tat-TAR complexes from the human and bovine immunodeficiency viruses (HIV and BIV). The viral Tat proteins are essential transcription elongation factors that bind to their respective TAR RNA hairpins located at the 5′-end of the nascent transcripts (12).…”

mentioning

confidence: 99%

Localized Influence of 2‘-Hydroxyl Groups and Helix Geometry on Protein Recognition in the RNA Major Groove

2005

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The local geometry of a DNA helix can influence protein recognition, but the sequencespecific features that contribute to helix structure are not fully understood, and even less is known about how RNA helix geometry may affect protein recognition. To begin to understand how local or global helix structure may influence binding in an RNA model system, we generated a series of DNA analogues of HIV and BIV TAR RNAs in which ribose sugars were systematically substituted in and around the known binding sites for argininamide and a BIV Tat arginine-rich peptide, respectively, and measured their corresponding binding affinities. For each TAR interaction, binding occurs in the RNA major groove with high specificity, whereas binding to the all-DNA analogue is weak and nonspecific. Relatively few substitutions are needed to convert either DNA analogue of TAR into a high-affinity binder, with the ribose requirements being restricted largely to regions that directly contact the ligand. Substitutions at individual positions show up to 70-fold differences in binding affinity, even at adjacent base pairs, while two base pairs at the core of the BIV Tat peptide-RNA interface are largely unaffected by deoxyribose substitution. These results suggest that the helix geometries and unique conformational features required for binding are established locally and are relatively insulated from effects more than one base pair away. It seems plausible that arginine-rich peptides are able to adapt to a mosaic helical architecture in which segments as small as single base steps may be considered as modular recognition units.

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Identification of Amino Acids that Promote Specific and Rigid TAR RNA-Tat Protein Complex Formation

Edwards

Robinson

Sigurdsson

2005

Chemistry & Biology

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The Tat protein and the transactivation responsive (TAR) RNA form an essential complex in the HIV lifecycle, and mutations in the basic region of the Tat protein alter this RNA-protein molecular recognition. Here, EPR spectroscopy was used to identify amino acids, flanking an essential arginine of the Tat protein, which contribute to specific and rigid TAR-Tat complex formation by monitoring changes in the mobility of nitroxide spin-labeled TAR RNA nucleotides upon binding. Arginine to lysine N-terminal mutations did not affect TAR RNA interfacial dynamics. In contrast, C-terminal point mutations, R56 in particular, affected the mobility of nucleotides U23 and U38, which are involved in a base-triple interaction in the complex. This report highlights the role of dynamics in specific molecular complex formation and demonstrates the ability of EPR spectroscopy to study interfacial dynamics of macromolecular complexes.

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Replication of Human Immunodeficiency Viruses Engineered with Heterologous Tat-Transactivation Response Element Interactions

Abstract: Human immunodeficiency viruses (HIVs) and the related bovine lentiviruses bovine immunodeficiency virus (BIV) and

Cited by 14 publications

References 58 publications

Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System

Selection of TAR RNA-Binding Chameleon Peptides by Using a Retroviral Replication System

Localized Influence of 2‘-Hydroxyl Groups and Helix Geometry on Protein Recognition in the RNA Major Groove

Identification of Amino Acids that Promote Specific and Rigid TAR RNA-Tat Protein Complex Formation

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