Structural basis for the specificity of the initiation of HIV-1 reverse transcription.
(1/289)
Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV-1 RNA and tRNA3Lys, but remains poorly understood at the three-dimensional level. We used chemical probing to gain insight into the three-dimensional structure of the viral RNA-tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three-dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA-tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3' end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure. (+info)
Single atom modification (O-->S) of tRNA confers ribosome binding.
(2/289)
Escherichia coli tRNALysSUU, as well as human tRNALys3SUU, has 2-thiouridine derivatives at wobble position 34 (s2U*34). Unlike the native tRNALysSUU, the full-length, unmodified transcript of human tRNALys3UUU and the unmodified tRNALys3UUU anticodon stem/loop (ASLLys3UUU) did not bind AAA- or AAG-programmed ribosomes. In contrast, the completely unmodified yeast tRNAPhe anticodon stem/loop (ASLPheGAA) had an affinity (Kd = 136+/-49 nM) similar to that of native yeast tRNAPheGmAA (Kd = 103+/-19 nM). We have found that the single, site-specific substitution of s2U34 for U34 to produce the modified ASLLysSUU was sufficient to restore ribosomal binding. The modified ASLLysSUU bound the ribosome with an affinity (Kd = 176+/-62 nM) comparable to that of native tRNALysSUU (Kd = 70+/-7 nM). Furthermore, in binding to the ribosome, the modified ASLLys3SUU produced the same 16S P-site tRNA footprint as did native E. coli tRNALysSUU, yeast tRNAPheGmAA, and the unmodified ASLPheGAA. The unmodified ASLLys3UUU had no footprint at all. Investigations of thermal stability and structure monitored by UV spectroscopy and NMR showed that the dynamic conformation of the loop of modified ASLLys3SUU was different from that of the unmodified ASLLysUUU, whereas the stems were isomorphous. Based on these and other data, we conclude that s2U34 in tRNALysSUU and in other s2U34-containing tRNAs is critical for generating an anticodon conformation that leads to effective codon interaction in all organisms. This is the first example of a single atom substitution (U34-->s2U34) that confers the property of ribosomal binding on an otherwise inactive tRNA. (+info)
Design, characterization and testing of tRNA3Lys-based hammerhead ribozymes.
(3/289)
A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3Lys without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3Lys-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy. (+info)
Coordination of tRNA nuclear export with processing of tRNA.
(4/289)
Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding. (+info)
Identification of a human immunodeficiency virus type 1 that stably uses tRNALys1,2 rather than tRNALys,3 for initiation of reverse transcription.
(5/289)
HIV-1 virions contain approximately equal amounts of tRNALys,3 and tRNALys1,2, yet tRNALys,3 has been found to be exclusively used for initiation of reverse transcription. Since previous studies have shown that even if the primer binding site (PBS) was mutated to be complementary to tRNALys1,2, the virus did not stably use tRNALys1,2 to initiate reverse transcription, the virus must have evolved a mechanism for the exclusive use of tRNALys,3 to initiate reverse transcription. To investigate how HIV-1 discriminates tRNALys1,2 from tRNALys,3 for initiation of reverse transcription, two proviral genomes that contain nucleotide changes in U5 and a PBS to be complementary to regions of tRNALys1,2 were constructed. One genome contains 5 [HXB2(L12-AC)] nucleotides while another contains 15 [HXB2(L12-ACgg)] nucleotides in U5 complementary to the anticodon region of tRNALys1,2. Viruses derived from the transfection of the proviral genomes were infectious in SupT1 cells. Analysis of the endogenous reverse transcription reactions from viruses derived from HXB2 (L12-AC) and HXB2 (L12-ACgg) obtained from transfection revealed that both exclusively used tRNALys1,2 to initiate reverse transcription. Following extensive in vitro culture, though, sequence analysis of proviral genomes revealed that while the virus derived from HXB2(L12-AC) stably maintained a PBS complementary to tRNALys1,2, the virus derived from HXB2 (L12-ACgg) had reverted back to contain a PBS complementary to tRNALys,3. RNA modeling of the U5-PBS of the genome from HXB2(L12-AC) supports the conclusion that the fine specificity for discrimination between tRNALys,3 and tRNALys1,2 for use as a primer for HIV-1 reverse transcription resides in the structure of the U5-PBS region of the viral genome. (+info)
Molecular requirements for human immunodeficiency virus type 1 plus-strand transfer: analysis in reconstituted and endogenous reverse transcription systems.
(6/289)
We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3' end of tRNA3Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3' rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3'-terminal bases by RNase H (added either in cis or trans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC. (+info)
Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses.
(7/289)
Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta-chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant. (+info)
Forty million years of mutualism: evidence for eocene origin of the yucca-yucca moth association.
(8/289)
The obligate mutualism between yuccas and yucca moths is a major model system for the study of coevolving species interactions. Exploration of the processes that have generated current diversity and associations within this mutualism requires robust phylogenies and timelines for both moths and yuccas. Here we establish a molecular clock for the moths based on mtDNA and use it to estimate the time of major life history events within the yucca moths. Colonization of yuccas had occurred by 41.5 +/- 9.8 million years ago (Mya), with rapid life history diversification and the emergence of pollinators within 0-6 My after yucca colonization. A subsequent burst of diversification 3.2 +/- 1.8 Mya coincided with evolution of arid habitats in western North America. Derived nonpollinating cheater yucca moths evolved 1.26 +/- 0.96 Mya. The estimated age of the moths far predates the host fossil record, but is consistent with suggested host age based on paleobotanical, climatological, biogeographical, and geological data, and a tentative estimation from an rbcL-based molecular clock for yuccas. The moth data are used to establish three alternative scenarios of how the moths and plants have coevolved. They yield specific predictions that can be tested once a robust plant phylogeny becomes available. (+info)