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The structure of the human tRNALys3 anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs.

Bilbille Y, Vendeix FA, Guenther R, Malkiewicz A, Ariza X, Vilarrasa J, Agris PF - Nucleic Acids Res. (2009)

Bottom Line: The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs.The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter.The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.

ABSTRACT
Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

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Sequence and secondary structure of the tRNALys3 interaction with Loop I of the HIV genome. (A) Sequence and secondary structure of human tRNALys3. The 3′-terminal, 18 nucleoside sequence of tRNALys3 (in red) is complementary to the HIV-1 Primer Binding Site, PBS. Residues in blue are complementary to the HIV-1 Primer Activation Signal, PAS. In purple are the residues of the anticodon domain fragment, ADF, studied in the article. In order not to have frayed and weak termini to the dodecamer duplex, a C43 was substituted for A43 to constitute a terminal C43•G158 base pair. The native post-transcriptional modifications are indicated according to standard nomenclature (80). (B) Chemical structures of the modified nucleosides in the ADF of human tRNALys3. The natural modification occurring at position 34 is 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) and that at position 37 is 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37). The uridine at position 39 is modified to pseudouridine, Ψ39. The modifications studied here were 2-thiouridine (s2U34) and pseudouridine, Ψ39. (C) The sequence and secondary structure of the A-rich Loop I of HIV-1 (residues 157–173). The residues in green (158–169) are those residues studied in this article. Residues A158 and C169 were changed to G158 and G159 in order that the dodecamer duplex have terminal G•C base pairs. (D) Duplexes composed of a fragment of the tRNALys3 anticodon domain, the ADF, and the A-rich Loop I of HIV-1, subtype G (VGL). Constructs included the individual and doubly modified ADFs with 2-thiouridine, s2U34 (purple) and pseudouridine, Ψ39 (blue). An additional construct of the ADF was substituted at positions 38 and 39 to create a full complement to the VGL (orange). The labeled pyrimidine nucleosides, [15N3]-uridine and 4-amino-[15NH2]-cytidine, are designated (green). The HIV-1 subtype B construct had a sequence with two Gs substituting for A166 and A167 (gray).
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Figure 1: Sequence and secondary structure of the tRNALys3 interaction with Loop I of the HIV genome. (A) Sequence and secondary structure of human tRNALys3. The 3′-terminal, 18 nucleoside sequence of tRNALys3 (in red) is complementary to the HIV-1 Primer Binding Site, PBS. Residues in blue are complementary to the HIV-1 Primer Activation Signal, PAS. In purple are the residues of the anticodon domain fragment, ADF, studied in the article. In order not to have frayed and weak termini to the dodecamer duplex, a C43 was substituted for A43 to constitute a terminal C43•G158 base pair. The native post-transcriptional modifications are indicated according to standard nomenclature (80). (B) Chemical structures of the modified nucleosides in the ADF of human tRNALys3. The natural modification occurring at position 34 is 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) and that at position 37 is 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37). The uridine at position 39 is modified to pseudouridine, Ψ39. The modifications studied here were 2-thiouridine (s2U34) and pseudouridine, Ψ39. (C) The sequence and secondary structure of the A-rich Loop I of HIV-1 (residues 157–173). The residues in green (158–169) are those residues studied in this article. Residues A158 and C169 were changed to G158 and G159 in order that the dodecamer duplex have terminal G•C base pairs. (D) Duplexes composed of a fragment of the tRNALys3 anticodon domain, the ADF, and the A-rich Loop I of HIV-1, subtype G (VGL). Constructs included the individual and doubly modified ADFs with 2-thiouridine, s2U34 (purple) and pseudouridine, Ψ39 (blue). An additional construct of the ADF was substituted at positions 38 and 39 to create a full complement to the VGL (orange). The labeled pyrimidine nucleosides, [15N3]-uridine and 4-amino-[15NH2]-cytidine, are designated (green). The HIV-1 subtype B construct had a sequence with two Gs substituting for A166 and A167 (gray).

Mentions: Retrovirions are packaged with an enrichment of a host cell tRNA for the purpose of priming reverse transcription. Human immunodeficiency virus type 1, HIV-1 and all other lentiviruses, evolved to select and recruit the cytoplasmic tRNALys3 and the other tRNALys isoacceptors, tRNALys1 and tRNALys2 (1). While the function of tRNALys1,2 in HIV-1 is not known, the selective packaging of HIV-1 reverse transcriptase primer tRNALys3 is required for optimizing both the annealing of tRNALys3 to viral RNA and the infectivity of the HIV-1 population (2). A considerable number of interactions are believed to occur between the human tRNALys3 and HIV-1, as determined by chemical and enzymatic probes (3). Eighteen nucleosides of the tRNA's 3′-terminal sequence (Figure 1A), part of the amino-acid accepting stem (nucleosides 66–76) and the 3′-side of the TΨC domain (tRNA nucleosides 59–65) (4), are bound to a complementary sequence in the viral RNA, the Primer Binding Sequence (PBS) (viral nucleosides 183–200), while the TΨC domain interacts with the Primer Activation Sequence (PAS) (5) (Figure 1A). The uridine-rich anticodon and adjacent nucleosides are bound to the adenosine-rich Loop I of the viral RNA (Figure 1A and C) (4). Deletion of the A-rich Loop I of HIV-1 that is complementary to the uridine-rich anticodon domain resulted in significantly reduced levels of infectivity and reduced synthesis of viral RNA (6–8). Constitution of a virus that was able to use a host cell tRNA other than tRNALys3 required not only the conversion of the PBS to the complement of the investigator-selected tRNA, but also the mutation of the AAAA sequence of HIV-1 Loop I to complement the anticodon sequence of the new tRNA (9–11). Changing one or the other of these sequences was not sufficient for maintaining the new interaction during prolonged culturing. Binding of the tRNA's anticodon region to the A-rich Loop I region has been associated with transition of viral replication from initiation to the highly processive elongation with a possible requirement for the tRNA's post-transcriptional modifications (12).Figure 1.


The structure of the human tRNALys3 anticodon bound to the HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs.

Bilbille Y, Vendeix FA, Guenther R, Malkiewicz A, Ariza X, Vilarrasa J, Agris PF - Nucleic Acids Res. (2009)

Sequence and secondary structure of the tRNALys3 interaction with Loop I of the HIV genome. (A) Sequence and secondary structure of human tRNALys3. The 3′-terminal, 18 nucleoside sequence of tRNALys3 (in red) is complementary to the HIV-1 Primer Binding Site, PBS. Residues in blue are complementary to the HIV-1 Primer Activation Signal, PAS. In purple are the residues of the anticodon domain fragment, ADF, studied in the article. In order not to have frayed and weak termini to the dodecamer duplex, a C43 was substituted for A43 to constitute a terminal C43•G158 base pair. The native post-transcriptional modifications are indicated according to standard nomenclature (80). (B) Chemical structures of the modified nucleosides in the ADF of human tRNALys3. The natural modification occurring at position 34 is 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) and that at position 37 is 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37). The uridine at position 39 is modified to pseudouridine, Ψ39. The modifications studied here were 2-thiouridine (s2U34) and pseudouridine, Ψ39. (C) The sequence and secondary structure of the A-rich Loop I of HIV-1 (residues 157–173). The residues in green (158–169) are those residues studied in this article. Residues A158 and C169 were changed to G158 and G159 in order that the dodecamer duplex have terminal G•C base pairs. (D) Duplexes composed of a fragment of the tRNALys3 anticodon domain, the ADF, and the A-rich Loop I of HIV-1, subtype G (VGL). Constructs included the individual and doubly modified ADFs with 2-thiouridine, s2U34 (purple) and pseudouridine, Ψ39 (blue). An additional construct of the ADF was substituted at positions 38 and 39 to create a full complement to the VGL (orange). The labeled pyrimidine nucleosides, [15N3]-uridine and 4-amino-[15NH2]-cytidine, are designated (green). The HIV-1 subtype B construct had a sequence with two Gs substituting for A166 and A167 (gray).
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Related In: Results  -  Collection

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Figure 1: Sequence and secondary structure of the tRNALys3 interaction with Loop I of the HIV genome. (A) Sequence and secondary structure of human tRNALys3. The 3′-terminal, 18 nucleoside sequence of tRNALys3 (in red) is complementary to the HIV-1 Primer Binding Site, PBS. Residues in blue are complementary to the HIV-1 Primer Activation Signal, PAS. In purple are the residues of the anticodon domain fragment, ADF, studied in the article. In order not to have frayed and weak termini to the dodecamer duplex, a C43 was substituted for A43 to constitute a terminal C43•G158 base pair. The native post-transcriptional modifications are indicated according to standard nomenclature (80). (B) Chemical structures of the modified nucleosides in the ADF of human tRNALys3. The natural modification occurring at position 34 is 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) and that at position 37 is 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37). The uridine at position 39 is modified to pseudouridine, Ψ39. The modifications studied here were 2-thiouridine (s2U34) and pseudouridine, Ψ39. (C) The sequence and secondary structure of the A-rich Loop I of HIV-1 (residues 157–173). The residues in green (158–169) are those residues studied in this article. Residues A158 and C169 were changed to G158 and G159 in order that the dodecamer duplex have terminal G•C base pairs. (D) Duplexes composed of a fragment of the tRNALys3 anticodon domain, the ADF, and the A-rich Loop I of HIV-1, subtype G (VGL). Constructs included the individual and doubly modified ADFs with 2-thiouridine, s2U34 (purple) and pseudouridine, Ψ39 (blue). An additional construct of the ADF was substituted at positions 38 and 39 to create a full complement to the VGL (orange). The labeled pyrimidine nucleosides, [15N3]-uridine and 4-amino-[15NH2]-cytidine, are designated (green). The HIV-1 subtype B construct had a sequence with two Gs substituting for A166 and A167 (gray).
Mentions: Retrovirions are packaged with an enrichment of a host cell tRNA for the purpose of priming reverse transcription. Human immunodeficiency virus type 1, HIV-1 and all other lentiviruses, evolved to select and recruit the cytoplasmic tRNALys3 and the other tRNALys isoacceptors, tRNALys1 and tRNALys2 (1). While the function of tRNALys1,2 in HIV-1 is not known, the selective packaging of HIV-1 reverse transcriptase primer tRNALys3 is required for optimizing both the annealing of tRNALys3 to viral RNA and the infectivity of the HIV-1 population (2). A considerable number of interactions are believed to occur between the human tRNALys3 and HIV-1, as determined by chemical and enzymatic probes (3). Eighteen nucleosides of the tRNA's 3′-terminal sequence (Figure 1A), part of the amino-acid accepting stem (nucleosides 66–76) and the 3′-side of the TΨC domain (tRNA nucleosides 59–65) (4), are bound to a complementary sequence in the viral RNA, the Primer Binding Sequence (PBS) (viral nucleosides 183–200), while the TΨC domain interacts with the Primer Activation Sequence (PAS) (5) (Figure 1A). The uridine-rich anticodon and adjacent nucleosides are bound to the adenosine-rich Loop I of the viral RNA (Figure 1A and C) (4). Deletion of the A-rich Loop I of HIV-1 that is complementary to the uridine-rich anticodon domain resulted in significantly reduced levels of infectivity and reduced synthesis of viral RNA (6–8). Constitution of a virus that was able to use a host cell tRNA other than tRNALys3 required not only the conversion of the PBS to the complement of the investigator-selected tRNA, but also the mutation of the AAAA sequence of HIV-1 Loop I to complement the anticodon sequence of the new tRNA (9–11). Changing one or the other of these sequences was not sufficient for maintaining the new interaction during prolonged culturing. Binding of the tRNA's anticodon region to the A-rich Loop I region has been associated with transition of viral replication from initiation to the highly processive elongation with a possible requirement for the tRNA's post-transcriptional modifications (12).Figure 1.

Bottom Line: The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs.The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter.The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.

ABSTRACT
Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

Show MeSH
Related in: MedlinePlus