Jennifer T. Miller

Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation.

Hundreds of structures of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid–RT structures including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. Thus, the hybrid has ready access to the RNase-H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states.

Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity.

High-throughput screening of National Cancer Institute libraries of synthetic and natural compounds identified the vinylogous ureas 2-amino-5,6,7,8-tetrahydro-4 H-cyclohepta[ b]thiophene-3-carboxamide (NSC727447) and N-[3-(aminocarbonyl)-4,5-dimethyl-2-thienyl]-2-furancarboxamide (NSC727448) as inhibitors of the ribonuclease H (RNase H) activity of HIV-1 and HIV-2 reverse transcriptase (RT). A Yonetani-Theorell analysis demonstrated that NSC727447, and the active-site hydroxytropolone RNase H inhibitor beta-thujaplicinol were mutually exclusive in their interaction with the RNase H domain. Mass spectrometric protein footprinting of the NSC727447 binding site indicated that residues Cys280 and Lys281 in helix I of the thumb subdomain of p51 were affected by ligand binding. Although DNA polymerase and pyrophosphorolysis activities of HIV-1 RT were less sensitive to inhibition by NSC727447, protein footprinting indicated that NSC727447 occupied the equivalent region of the p66 thumb. Site-directed mutagenesis using reconstituted p66/p51 heterodimers substituted with natural or non-natural amino acids indicates that altering the p66 RNase H primer grip significantly affects inhibitor sensitivity. NSC727447 thus represents a novel class of RNase H antagonists with a mechanism of action differing from active site, divalent metal-chelating inhibitors that have been reported.

Mechanisms for Priming DNA Synthesis

DNA replication is a semiconservative process in which a DNA polymerase uses one DNA strand as a template for the synthesis of a second, complementary, DNA strand. However, in contrast to RNA polymerases, which can initiate RNA synthesis on a DNA template de novo, all DNA polymerases require a preexisting primer on which to initiate DNA synthesis (Kornberg and Baker 1992). One apparent exception to this rule is a mitochondrial DNA (mtDNA)-encoded reverse transcriptase (RT) in Neurospora (Wang and Lambowitz 1993). Preexisting primers can be classified into four groups. The simplest primer consists of the 3′-hydroxyl (3′-OH) termini of DNA chains that are complementary to the DNA template and thereby form a stable duplex structure at the site where DNA synthesis begins. This primer is used for DNA repair (Friedberg and Wood, this volume), parvovirus DNA replication (Brush and Kelly; Cotmore and Tattersall; both this volume), some RTs. The second type of primer consists of a deoxyribonucleoside monophosphate that is covalently attached to a specific serine, threonine, or tyrosine residue of a protein. Examples are bacteriophage, plasmids, and animal viruses that replicate as a linear DNA genome, and animal viruses such as hepadnaviruses whose genome is partially double-stranded and partially single-stranded. The third type of primer consists of tRNA molecules that anneal to specific sequences in the RNA genomes of retroviruses where their 3′-OH termini are utilized by RT. The fourth class of primers consists of nascent RNA chains. These comprise nascent RNA transcripts that are processed to create a...

Human Immunodeficiency Virus Type 2 Reverse Transcriptase Activity in Model Systems That Mimic Steps in Reverse Transcription

ABSTRACT Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.

Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry

Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)–containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR retrotransposon Ty3 in the presence of its polypurine tract–containing RNA-DNA hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture whose assembly is substrate dependent. Moreover, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer.

Interaction Between Reverse Transcriptase and Integrase is Required for Reverse Transcription During HIV-1 Replication

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication requires reverse transcription of its RNA genome into a double-stranded cDNA copy, which is then integrated into the host cell chromosome. The essential steps of reverse transcription and integration are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), respectively. In vitro, HIV-1 RT can bind with IN, and the C-terminal domain (CTD) of IN is necessary and sufficient for this binding. To better define the RT-IN interaction, we performed nuclear magnetic resonance (NMR) spectroscopy experiments to map a binding surface on the IN CTD in the presence of RT prebound to a duplex DNA construct that mimics the primer-binding site in the HIV-1 genome. To determine the biological significance of the RT-IN interaction during viral replication, we used the NMR chemical shift mapping information as a guide to introduce single amino acid substitutions of nine different residues on the putative RT-binding surface in the IN CTD. We found that six viral clones bearing such IN substitutions (R231E, W243E, G247E, A248E, V250E, and I251E) were noninfectious. Further analyses of the replication-defective IN mutants indicated that the block in replication took place specifically during early reverse transcription. The recombinant INs purified from these mutants, though retaining enzymatic activities, had diminished ability to bind RT in a cosedimentation assay. The results indicate that the RT-IN interaction is functionally relevant during the reverse transcription step of the HIV-1 life cycle. IMPORTANCE To establish a productive infection, human immunodeficiency virus type 1 (HIV-1) needs to reverse transcribe its RNA genome to create a double-stranded DNA copy and then integrate this viral DNA genome into the chromosome of the host cell. These two essential steps are catalyzed by the HIV-1 enzymes reverse transcriptase (RT) and integrase (IN), respectively. We have shown previously that IN physically interacts with RT, but the importance of this interaction during HIV-1 replication has not been fully characterized. In this study, we have established the biological significance of the HIV-1 RT-IN interaction during the viral life cycle by demonstrating that altering the RT-binding surface on IN disrupts both reverse transcription and viral replication. These findings contribute to our understanding of the RT-IN binding mechanism, as well as indicate that the RT-IN interaction can be exploited as a new antiviral drug target.

Reverse Transcription in the Saccharomyces cerevisiae Long-Terminal Repeat Retrotransposon Ty3

Converting the single-stranded retroviral RNA into integration-competent double-stranded DNA is achieved through a multi-step process mediated by the virus-coded reverse transcriptase (RT). With the exception that it is restricted to an intracellular life cycle, replication of the Saccharomyces cerevisiae long terminal repeat (LTR)-retrotransposon Ty3 genome is guided by equivalent events that, while generally similar, show many unique and subtle differences relative to the retroviral counterparts. Until only recently, our knowledge of RT structure and function was guided by a vast body of literature on the human immunodeficiency virus (HIV) enzyme. Although the recently-solved structure of Ty3 RT in the presence of an RNA/DNA hybrid adds little in terms of novelty to the mechanistic basis underlying DNA polymerase and ribonuclease H activity, it highlights quite remarkable topological differences between retroviral and LTR-retrotransposon RTs. The theme of overall similarity but distinct differences extends to the priming mechanisms used by Ty3 RT to initiate (−) and (+) strand DNA synthesis. The unique structural organization of the retrotransposon enzyme and interaction with its nucleic acid substrates, with emphasis on polypurine tract (PPT)-primed initiation of (+) strand synthesis, is the subject of this review.

Novel insights from structural analysis of lentiviral and gammaretroviral reverse transcriptases in complex with RNA/DNA hybrids

Structures of HIV-1 reverse transcriptase (RT) have been reported in several forms, but only one contains an RNA/DNA hybrid, the conformation of which has been controversial. We have been successful in obtaining three structures of HIV-1 RT complexed with a non-nucleoside RT inhibitor (NNRTI) and an RNA/DNA hybrid [1]. In the presence of an NNRTI, our RNA/DNA structure differs from all prior nucleic acid bound to RT including the previously-reported RNA/DNA hybrid derived froom the polypurine tract. The enzyme structure observed in our cocrystals also differs from all previous RT-DNA complexes. As a result, the hybrid has ready access to the ribonuclease H (RNase H) active site. These observations collectively reinforce previous proposals that an RT-nucleic acid complex may be required to adopt independent structural states competent for DNA synthesis and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states. Structural features of the nucleoprotein complex, including drug resistance mutations, have been verified by site-directed mutagenesis, and will be presented. n nAlthough the single-subunit RT of Moloney murine leukemia virus (Mo-MLV) has been extensively characterized biochemically, structural information is lacking that describes the substrate binding mechanism for this RT species. We also present data on the first crystal structure of a complex between an RNA/DNA hybrid and the 72 kDa single-subunit RT from the related xenotropic murine leukemia virus-related virus (XMRV) [2]. A comparison of this structure with its HIV-1 counterpart shows that substrate binding around the DNA polymerase active site is conserved but differs between the two enzymes in their thumb and connection subdomains. Small-angle X-ray scattering (SAXS) was used to model full-length XMRV RT, demonstrating its flexible RNase H domain becomes ordered in the presence of substrate, a key difference between monomeric and dimeric RTs.

Ty3 reverse transcriptase complexed with an RNA/DNA hybrid shows structural and functional asymmetry

Retrotransposons are mobile genetic elements that replicate through an RNA intermediate. They are divided into two groups, depending on the presence of flanking long-terminal repeat (LTR) sequences. Approximately 40% of the human genome is derived from retroelements with 8% corresponding to the LTR class. Retroviruses, such as human immunodeficiency virus (HIV-1) evolved from LTR elements through acquiring the envelope genes which allow them to leave the cell and infect other cells. Reverse transcriptase (RT) is a critical enzyme of retroelements, combining DNA polymerase and RNase H activities to convert the (+) strand RNA genome into double-stranded DNA that is competent for integration. However, In contrast to the extensive structural characterization of retroviral RTs, detailed information on the enzymes from LTR-containing retrotransposons such as Ty3 of Saccharomyces cerevisiae, is lacking. n nTy3 belongs to the Gypsy family and its RT is perhaps the most extensively characterized LTR-retrotransposon enzyme with respect to its catalytic activities, as well as the nucleic acid duplexes with which it interacts. Although structural motifs ascribed to substrate recognition and catalysis are generally similar to those of vertebrate retroviral RTs, a notable difference is replacement of the highly-conserved active site His with Tyr in the RNase H domain of Ty3 RT. More significantly Ty3 RT lacks the connection, or tether, between its DNA polymerase and RNase H active sites, with the consequence that on the nucleic acid substrate they are separated by ~13 bp as opposed to the 17-18 bp observed for lentiviral and gammaretroviral enzymes. Structural similarity between the HIV-1 connection and its RNase H domain originally suggested the latter arose through domain duplication, while an alternative theory proposes the functional RNase H domain was acquired as a new, and more efficient element from a source outside the LTR retrotransposons. Structural and biochemical information on subdomain organization of Ty3 RT is therefore important in understanding the evolutionary divergence between these essential vertebrate retroviral and LTR-retrotransposon enzymes. To this end, we provide the first structure of Ty3 RT in complex with an RNA/DNA hybrid at 3.1 A resolution. The fully-active enzyme is an asymmetric homodimer of 55 kDa subunits, and is formed only in the presence of the RNA/DNA substrate. Modeling the spatial separation between the DNA polymerase and RNase H active sites, coupled with site-directed mutagenesis, suggests that one active site of each subunit contributes enzyme activity.

Reply to [ldquo]Structural requirements for RNA degradation by HIV-1 reverse transcriptase[rdquo]

In their correspondence, Das and colleagues 1 raise a number of issues about our work, which we address below. n nFirstly, we have not claimed that the RNA scissile phosphate is situated in the RNase H active site of HIV-1 RT ready for cleavage. Instead we reported 2 that our structures are “compatible with RNA degradation” (not “catalytically relevant” as incorrectly quoted by Das et al. 1), whereas all previous RT-nucleic acid (NA) complex structures are incompatible with RNA degradation. The incompatibility with RNA cleavage in the previous RT-NA complexes lies in the orientation of the NA substrate, not in the distance between NA and the RNase H active site, as we clearly demonstrated in Figure S5 2. In the previous RT-NA complexes, one DNA strand can be connected with the RNA/DNA hybrid positioned for RNA cleavage (Fig. 1a) according to the human and bacterial RNase H1-RNA/DNA hybrid structure 3,4, but the second strand (RNA equivalent) cannot be connected because of a 14 A gap. This gap cannot be closed by any amount of bending or unwinding of the duplex 3. In the three RT-RNA/DNA-NNRTI complex structures we reported 2, the RNA/DNA hybrids are oriented such that there is no long a gap (Fig. 1b), and a slight adjustment of the RNA strand would permit hydrolysis as illustrated in Figures 5 2. Indeed, the nearest RNA phosphate in our structures is 8.8 A from the active site carboxylate as depicted in Figure 5 2. We should clarify that the distance between the scissile phosphate and the active site in the human RNase H1-RNA/DNA complex is 4.6A (PDB: 2Q39) 3. Thus the scissile phosphate in the 4B3O structure needs to move 4.2A to be positioned for cleavage. It is not the distance but rather the NA orientation that makes our RNA/DNA hybrid compatible with RNA degradation, which is also evident in Fig. 1a of the correspondence by Das and colleague 1. n n n nFigure 1 n nDifferences between previous RT-NA complexes and our RT-RNA/DNA structures. (a) All RT-NA structrues previously reported are similar 2 and represented here by the RT-DNA-dATP ternary complex (PDB:3KK2 6) in the polymerization mode. Human RNase H1-RNA/DNA structure ... n n n nSecondly, we disagree with the statement that our structure (4B3O) is most closely related to the RT-DNA/DNA-nevirapine structure (PDB 3V8I)5. Both the protein and nucleic acid of the 3V8I structure are more similar to that of RT-DNA complexes than to our RT-RNA/DNA hybrid complexes, as we showed in Figure 3 2. For example, the RNA/DNA hybrid in our structure has the A-form conformation, while the DNA duplex in 3V8I as well as all previously reported RT-NA complexes are largely B-form 2 (Fig. 1). n nThirdly, with regard to the alleged crystal packing effects, we had noticed the lattice contact all along but found it irrelevant to RT-RNA/DNA hybrid complex formation, based on three different crystal forms of RT-RNA/DNA hybrid complexes (Figures 1 and S1 2). The region near the p66 thumb domain, with which Das and colleagues are concerned 1, is not involved in crystal packing in at least one of our three crystal structures 2, and the A-form conformation of our entire RNA/DNA is independent of this lattice contact. n nFourthly, on the issue of the nick in the RNA strand used in one of the three RT-RNA/DNA complex structures presented in our paper, the nick was engineered by design as reported in the main text and Methods section, and depicted in Figures 1 and S1 2. However, the other two RT-RNA/DNA complex structures (PDB: 4B3P and 4B3Q) contain a continuous RNA strand 2. The statement by Das and colleagues that a continuous RNA/DNA duplex would not be able to adopt the conformation or trajectory adopted by the nicked hybrid 1 is simply incorrect. The RNA/DNA hybrids in all three of our structures, including two with a continuous RNA strand are in similar conformations (Figures 1d and S3 2). n nFinally, regarding cross-linking between RT and nucleic acid in previous RT complexes, we have not questioned the validity of this strategy in capturing HIV-1 RT in the DNA polymerization mode. Rather, we respectfully pointed out that in the more than 20 RT-DNA crosslinked structures, the DNAs are all in a similar conformation, and one that is incompatible with RNA degradation.volume 20 number 12 DeCember 2013 nature structural & molecular biology Kalyan Das1, Stefan G. Sarafianos2 & Eddy Arnold1 1Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA. 2Christopher S. Bond Life Sciences Center, Department of Molecular Microbiology & Immunology and Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri, USA. e-mail: arnold@cabm.rutgers.edu