Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

Werner, Susanne; Wöhrl, Birgitta M.

doi:10.1128/jvi.74.7.3245-3252.2000

Cited by 17 publications

(27 citation statements)

References 37 publications

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“…The 55-kDa re-combinant form of the Ty3 RT is active on a synthetic DNA and RNA templates (48) and on a Ty3-like template with Ty3 NC and tRNA iMet primer for minus-strand, strong-stop DNA synthesis in vitro (11). Nonetheless, this does not exclude the possibility that the major replication-competent form of Ty3 RT is an ␣-␤-type heterodimer similar to avian leukosis-sarcoma (55). Ty3 VLPs do contain a 115-kDa RT-IN fusion protein, although in smaller amounts than the 55-kDa RT or the 61-kDa IN (31,35).…”

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confidence: 98%

Ty3 Integrase Is Required for Initiation of Reverse Transcription

Nymark-McMahon

Beliakova-Bethell

Darlix

et al. 2002

J Virol

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Ty3 is a 5.4-kb, retroviruslike element of the yeast Saccharomyces cerevisiae (49). The two open reading frames of Ty3, GAG3 and POL3, are analogous to the retroviral gag and pol genes. They encode the structural proteins capsid (CA) and nucleocapsid (NC) and the enzymes protease (PR), reverse transcriptase (RT), and integrase (IN), respectively (29)(30)(31)35). These proteins, associated with genomic RNA, assemble into viruslike particles (VLPs), and polyprotein maturation and reverse transcription ensue. Ty3 differs, however, from retroviruses in that it inserts specifically into the transcription initiation site of genes transcribed by RNA polymerase III (8). In addition, Ty3 IN has extended, weakly conserved domains amino and carboxyl terminal to the central, conserved domain that contains the zinc-binding motif and catalytic D-D-(35)E triad. These terminal domains were previously examined by molecular genetics in order to understand their role(s) in functions unique to Ty3. Deletion (37) and alanine-scanning mutagenesis (47) revealed pleiotropic effects of mutations in the nonconserved IN domains on Ty3 replication, including effects on RT and IN protein processing and stability, DNA content of the VLP, 3Ј end processing, and nuclear entry. Mutations that eliminate large portions of the human immunodeficiency virus type 1 (HIV-1) IN-coding sequence and point mutations in the zinc finger and catalytic core region (F185Y) result in lower amounts of DNA circles in infected cells (17,18,45). More recently, these mutations and a small carboxyl-terminal deletion of IN were shown to specifically reduce the amount of early reverse transcription intermediates that could be used as templates in a PCR to amplify the R-U5 region (58). In another study, nine conserved S, T, Y, K, and R residues of IN were mutated to test for contributions to reverse transcription. R186, which maps near the nuclear localization sequence of HIV-1 IN (4), was required for wild-type amounts of an early replication intermediate as measured by PCR (53). Consistent with these effects of IN structure on RT function, HIV-1 IN and RT have been shown to interact (58). Recently, similar findings have been reported for the effects of mutations in Moloney murine leukemia virus IN on cDNA production (40), suggesting that the contribution of IN to reverse transcription may be a more general phenomenon. Nevertheless, the exact contribution of IN to reverse transcription is not yet understood at the molecular level.Models to explain the contribution of Ty3 IN to extrachromosomal Ty3 DNA would be compatible with effects at several different points. These include roles in initiation complex assembly, minus-strand or plus-strand transfers, elongation, and stabilization of the extrachromosomal DNA. In replication, the IN domain could function in cis to the RT domain, for example as part of an RT-IN fusion, or in trans, for example by folding RT. Precedents for these models can be found among the retroviruses. In the case of avian leukosis-sarcoma, in the ␣-␤...

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confidence: 98%

Ty3 Integrase Is Required for Initiation of Reverse Transcription

Nymark-McMahon

Beliakova-Bethell

Darlix

et al. 2002

J Virol

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“…Mutating the active-site residue Asp505 to Asn leads to RNase H-deficient enzymes (30). To examine the polymerization activities of the mutants and to analyze whether the presence of an active RNase H is required to polymerize products that are longer than the original template, we used the homopolymeric poly(rA)/oligo(dT) 16 as a substrate.…”

Section: Resultsmentioning

confidence: 99%

“…The methods for expressing and isolating wild-type and mutant RSV RT enzymes and the evaluation of RT enzyme activities have been described previously (29,30).…”

Section: Methodsmentioning

confidence: 99%

“…Three forms of RT have been isolated from avian leukosis and sarcoma viruses: a 63-kDa protein designated ␣ which contains the polymerase and RNase H domains; the ␤ protein, with an apparent molecular weight of 95 kDa, which in addition to the two RT domains harbors the IN domain; and the most abundant heterodimeric ␣␤ RT (11,14,15). We have shown previously that different forms of RSV RT can be expressed in and purified from insect cells using the baculovirus expression system (29,30).In this study we wanted to analyze the function of the different RSV enzymes in the specific minus-strand transfer reaction. To find out more about the function of the C-terminal 4.1-kDa protein of Pol, we also included the entire Pol protein and the heterodimeric ␣Pol RT in our studies.…”

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confidence: 99%

“…In order to determine the role of the RNase H activity for RSV strand transfer, two RNase H-deficient mutants which we characterized recently were also analyzed (30). In addition, we investigated the influence of the viral NC protein on the minusstrand transfer reaction.…”

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Requirements for Minus-Strand Transfer Catalyzed by Rous Sarcoma Virus Reverse Transcriptase

Werner

Vogel‐Bachmayr

Hollinderbäumer

et al. 2001

J Virol

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We have examined the specific minus-strand transfer reactions that occur after the synthesis of minus strong-stop DNA and nonspecific strand switching on homopolymeric poly(rA) templates with different types of Rous sarcoma virus (RSV) reverse transcriptases. Three different types of reverse transcriptases can be isolated from virions of RSV: heterodimeric ␣␤ and homodimeric ␣ and ␤. The mechanism of minus-strand transfer was examined using a model primer-template substrate corresponding to the 5-and 3-terminal RNA regions of the RSV genome. The results reveal that the RNase H activity of RSV reverse transcriptases is required for minus-strand transfer. Less than 2% of strand transfer of the extended product is detectable with RNase H-deficient enzymes. We could show that the ␣ homodimer lacking the integrase domain can perform strand transfer almost as efficiently as the ␣␤ and ␣Pol heterodimers. In contrast, the activities of ␤ and Pol for minus-strand transfer are reduced. Furthermore, a two-to fivefold increase in minus-strand transfer activities was observed in the presence of human immunodeficiency virus type 1 nucleocapsid protein.Reverse transcriptases (RTs) of retroviruses catalyze the synthesis of double-stranded DNA using the single-stranded viral genome as the template. Synthesis of the first DNA strand starts from a tRNA primer hybridized to the viral RNA at the primer-binding site complementary to the last 18 nucleotides of the tRNA. As the primer-binding site is located close to the 5Ј end of the viral RNA, DNA synthesis of the minus-strand DNA can only proceed after the DNA product is transferred to the 3Ј end of the RNA. This process is called minus-strand transfer. A second strand transfer event occurs during the synthesis of the plus-strand DNA (for a review, see reference 5).These strand transfer reactions are essential for the creation of the complete long terminal repeats (LTRs). LTRs are formed when the sequences close to the ends of the RNA are duplicated. The strand transfer reactions are specific processes. In contrast, the template-switching reactions that take place between the two copies of genomic RNA during polymerization are not sequence specific.Minus-strand transfer requires that identical direct repeats, designated R for repeated, be present at both ends of the RNA template. The length of these R regions varies in different retroviruses. R is 96 nucleotides long in human immunodeficiency virus (HIV) and includes the highly structured TAR region necessary for specific binding of the Tat transactivator protein. In contrast, R is only 21 nucleotides in length in Rous sarcoma virus (RSV) and 12 nucleotides in mouse mammary tumor virus, indicating that the R sequence is not highly conserved; rather, the major function of it appears to be to accept the transferring strand (5). In the case of HIV, it has been shown that minus-strand transfer is profoundly increased in the presence of the viral nucleocapsid (NC) protein (2,13,17,20). It has been suggested that the NC protein destabiliz...

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Small Molecule Inhibitors Targeting HIV‐1 Reverse Transcriptase Dimerization

et al. 2008

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The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t(1/2) of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.

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Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

Cited by 17 publications

References 37 publications

Ty3 Integrase Is Required for Initiation of Reverse Transcription

Ty3 Integrase Is Required for Initiation of Reverse Transcription

Requirements for Minus-Strand Transfer Catalyzed by Rous Sarcoma Virus Reverse Transcriptase

Small Molecule Inhibitors Targeting HIV‐1 Reverse Transcriptase Dimerization

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