Priming of HIV replication by tRNALys3: role of reverse transcriptase

Litvak, Simón; Sarih-Cottin, Leila; Fournier, Michel; Andréola, Marie‐Line; Tarragó-Litvak, Laura

doi:10.1016/0968-0004(94)90203-8

Cited by 40 publications

(90 citation statements)

References 27 publications

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“…In this process, the 3-end of tRNA 3 Lys must be annealed to the primer binding site of HIV-1 genomic RNA, and the two molecules together form a complex structure. During annealing, the nucleocapsid (NC) protein enhances the unwinding of tertiary structures within both RNA molecules.…”

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A HIV-1 Minimal Gag Protein Is Superior to Nucleocapsid at in Vitro tRNA3Lys Annealing and Exhibits Multimerization-induced Inhibition of Reverse Transcription

Roldán

Warren

Russell

et al. 2005

Journal of Biological Chemistry

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HIV-1 uses tRNA 3Lys to prime reverse transcription of its viral RNA. In this process, the 3-end of tRNA 3 Lys must be annealed to the primer binding site of HIV-1 genomic RNA, and the two molecules together form a complex structure. During annealing, the nucleocapsid (NC) protein enhances the unwinding of tertiary structures within both RNA molecules. Moreover, the packaging of tRNA 3Lys occurs prior to viral budding at a time when NC is still part of the Pr55Gag polyprotein. In contrast, Pr55Gag is able to produce virus-like particles on its own. We have recently shown that an N-terminal extended form of NC (mGag), containing all of the minimal elements required for virus-like particle formation, possesses greater affinity for HIV-1 genomic RNA than does NC alone. We have now studied the tRNA 3 Lys -annealing properties of mGag in comparison to those of NC and report that the former is more efficient in this regard than the latter. We have also tested each of a mutant version of mGag, an extended form of mGag, and an almost full-length form of Gag, and showed that all of these possessed greater tRNA-annealing capacity than did the viral NC protein. Yet, surprisingly, multimerization of Gag-related proteins did not abrogate this annealing process but rather resulted in dramatically reduced levels of reverse transcriptase processivity. These results suggest that the initial stages of reverse transcription may be regulated by the multimerization of Pr55Gag polyprotein at times prior to the cleavage of NC.

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confidence: 99%

A HIV-1 Minimal Gag Protein Is Superior to Nucleocapsid at in Vitro tRNA3Lys Annealing and Exhibits Multimerization-induced Inhibition of Reverse Transcription

Roldán

Warren

Russell

et al. 2005

Journal of Biological Chemistry

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“…These activities facilitate the conversion of single-stranded genomic RNA to the double-stranded proviral DNA which is integrated into the host genome. Synthesis of the first DNA strand, the minus strand DNA, is initiated from a cellular tRNA which binds with its 3Ј terminus to the complementary primer-binding site near the 5Ј-end of the viral RNA (2)(3)(4). The newly synthesized minus-strand DNA serves in turn as template for the synthesis of the second DNA strand or plusstrand.…”

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Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals

Götte¹,

Maier²,

Groß³

et al. 1998

Journal of Biological Chemistry

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Reverse transcriptase (RT)-2؉ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg 2؉ -dependent cleavage. The Fe 2؉ -dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3-end and the Fe 2؉ -dependent cleavage position at the RNase H active site. Retroviral reverse transcriptases (RTs)1 are multifunctional enzymes having an RNA-dependent and a DNA-dependent polymerase activity and a ribonuclease H (RNase H) activity, which degrades the RNA strand of RNA/DNA hybrids (1). These activities facilitate the conversion of single-stranded genomic RNA to the double-stranded proviral DNA which is integrated into the host genome. Synthesis of the first DNA strand, the minus strand DNA, is initiated from a cellular tRNA which binds with its 3Ј terminus to the complementary primer-binding site near the 5Ј-end of the viral RNA (2-4). The newly synthesized minus-strand DNA serves in turn as template for the synthesis of the second DNA strand or plusstrand. Prerequisite for the plus-strand synthesis is removal of the viral RNA from the minus-strand DNA. Both activities act simultaneously, presupposing a spatial and temporal interdependence of the active sites.The interplay of the two activities has been studied extensively in RT of human immunodeficiency virus type 1 (HIV-1) (5-13). It has been demonstrated that the viral RNA template is cleaved 18 nucleotides upstream with respect to the 3Ј-end of the nascent primer terminus (7) during minus-strand synthesis. But cleavage does not occur after each nucleotide incorporation step, indicating that the two activities, the polymerase and RNase H activity, are not coupled in a 1:1 mode (11-14). Gopalakrishnan et al. (7) suggested a temporal coordination of the two activities due to kinetic coupling. Cleavage can occur if the rate of DNA polymerization is lower than the rate of RNA hydrolysis. A similar concept might also explain the resistance of dsRNA toward cleavage. It has been shown that dsRNA formed by the tRNA and the viral RNA during initiation of minus-strand synthesis remains intact during DNA synthesis, but is cleaved, if the DNA synthesis is artificially stopped through use of chain terminating nucleotides (9). This can be explained by the fact that the rate of polymerization is higher than the rate of RNA template hydrolysis of dsRNA in line with the kinetic coupling model (9). Ribonuclease activity on dsRNA, which is believed to be mediated by t...

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“…HIV-1 RT is a heterodimer composed of equimolar amounts o f two polypeptides of 66 kDa and 51 kDa (for recent reviews on reverse transcriptase, see Skalka andGoff, 1993 andTarrago-Litvak et al, 1994). The sequence of the HIV-1-primer-binding site (Wain-Hobson et al, 1985) indicates that HTV-1 RT uses tRNA)Ys for the initiation of cDNA synthesis (for a review, see Litvak et al, 1994). Previously, we have analyzed the interaction of HIV-1 RT with synthetic templates and primers of different length and structure .…”

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High‐Affinity Interaction of Human Immunodeficiency Virus Type‐1 Reverse Transcriptase with Partially Complementary Primers

Zakharova

Tarragó-Litvak

Maksakova

et al. 1995

European Journal of Biochemistry

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The comparison of K,, and V,,,,, values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-I (HIV-1) reverse transcriptase was carried out. The primers were : (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and V,,,,,, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1 -1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT),(dCdG),(dT), showed higher affinity for the enzyme than (dT), or (dT), primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNAy', the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus. besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme. K~J N Y I~~T :human immunodeficiency virus type-1 reverse transcriptase ; mismatched primer elongation ; kinetic analysis ; discrimination ; tRNAiy' Studies of enzyme-kinetic mechanisms responsible for the accuracy of DNA replication are essential in understanding the molecular basis of mutagenesis. The ability of DNA polymera s e~ from bacteria, bacteriophages and eucaryotic organisms to copy DNA templates with high fidelity has been ascribed, in part, to the associated 3'-5' exonuclease proofreading activity of these DNA polymerases (Brutiag and Kornberg;Muzycska et al., 1972;Fry and Loeb, 1986). The human immunodeficiency virus type-1 (HIV-1) has been found to exhibit extensive penomic heterogeneity. An explanation for generating sequence diversity derives from the fact that reverse transcriptase (RT) lacks 3'-5' exonuclease proofreading activity for Corre.\L.sponrlence to G. A.

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Priming of HIV replication by tRNALys3: role of reverse transcriptase

Cited by 40 publications

References 27 publications

A HIV-1 Minimal Gag Protein Is Superior to Nucleocapsid at in Vitro tRNA3Lys Annealing and Exhibits Multimerization-induced Inhibition of Reverse Transcription

A HIV-1 Minimal Gag Protein Is Superior to Nucleocapsid at in Vitro tRNA3Lys Annealing and Exhibits Multimerization-induced Inhibition of Reverse Transcription

Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals

High‐Affinity Interaction of Human Immunodeficiency Virus Type‐1 Reverse Transcriptase with Partially Complementary Primers

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