Ajaykumar C. Vora

A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity.

Abstract A 32,000-dalton nucleic acid-binding protein (p32), possessing DNA endonuclease activity, has been identified in avian myeloblastosis virus (AMV) and Rous sarcoma virus (Prague B strain) cores. The p32 nucleic acid-binding protein was purified from AMV virions by phosphocellulose, poly (U)-Sepharose 4B, and poly(C)-agarose chromatography and glycerol gradient centrifugation. A DNA endonuclease activity was found associated with p32 throughout purification, but the protein possessed no detectable exonuclease activity with natural or synthetic DNA or RNA substrates. In the presence of Mg 2+ , the p32-associated DNA endonuclease activity was able to convert a variety of supercoiled DNA molecules to the relaxed form by introducing single-stranded nicks into the DNA. There was only one nick per supercoiled Escherichia coli ColE 1 DNA strand whether the molecular ratio of p32 to DNA was 12, 60, 130, or 260 to 1. Under the same reaction conditions, the p32-associated endonuclease activity was much less efficient in nicking single- or double-stranded linear DNA molecules. Alkaline sucrose gradient centrifugation analysis of ColE 1 DNA which was nicked by p32 and subsequently digested by EcoRI endonuclease suggested that a limited number of preferred regions for p32 endonuclease activity existed on this DNA. This site-specificity was lost in the presence of Mn 2+ , but the efficiency of the DNA endonuclease activity was increased approximately 10-fold.

Inhibition of Human Immunodeficiency Virus Type 1 Concerted Integration by Strand Transfer Inhibitors Which Recognize a Transient Structural Intermediate

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) integrase (IN) inserts the viral DNA genome into host chromosomes. Here, by native agarose gel electrophoresis, using recombinant IN with a blunt-ended viral DNA substrate, we identified the synaptic complex (SC), a transient early intermediate in the integration pathway. The SC consists of two donor ends juxtaposed by IN noncovalently. The DNA ends within the SC were minimally processed (∼15%). In a time-dependent manner, the SC associated with target DNA and progressed to the strand transfer complex (STC), the nucleoprotein product of concerted integration. In the STC, the two viral DNA ends are covalently attached to target and remain associated with IN. The diketo acid inhibitors and their analogs effectively inhibit HIV-1 replication by preventing integration in vivo. Strand transfer inhibitors L-870,810, L-870,812, and L-841,411, at low nM concentrations, effectively inhibited the concerted integration of viral DNA donor in vitro. The inhibitors, in a concentration-dependent manner, bound to IN within the SC and thereby blocked the docking onto target DNA, which thus prevented the formation of the STC. Although 3′-OH recessed donor efficiently formed the STC, reactions proceeding with this substrate exhibited marked resistance to the presence of inhibitor, requiring significantly higher concentrations for effective inhibition of all strand transfer products. These results suggest that binding of inhibitor to the SC occurs prior to, during, or immediately after 3′-OH processing. It follows that the IN-viral DNA complex is “trapped” by the strand transfer inhibitors via a transient intermediate within the cytoplasmic preintegration complex.

DNase Protection Analysis of Retrovirus Integrase at the Viral DNA Ends for Full-Site Integration In Vitro

ABSTRACT Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN–4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ∼20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.

Physical Trapping of HIV-1 Synaptic Complex by Different Structural Classes of Integrase Strand Transfer Inhibitors

Raltegravir is an FDA approved inhibitor directed against human immunodeficiency virus type 1 (HIV-1) integrase (IN). In this study, we investigated the mechanisms associated with multiple strand transfer inhibitors capable of inhibiting concerted integration by HIV-1 IN. The results show raltegravir, elvitegravir, MK-2048, RDS 1997, and RDS 2197 all appear to encompass a common inhibitory mechanism by modifying IN-viral DNA interactions. These structurally different inhibitors bind to and inactivate the synaptic complex, an intermediate in the concerted integration pathway in vitro. The inhibitors physically trap the synaptic complex, thereby preventing target DNA binding and thus concerted integration. The efficiency of a particular inhibitor to trap the synaptic complex observed on native agarose gels correlated with its potency for inhibiting the concerted integration reaction, defined by IC(50) values for each inhibitor. At low nanomolar concentrations (<50 nM), raltegravir displayed a time-dependent inhibition of concerted integration, a property associated with slow-binding inhibitors. Studies of raltegravir-resistant IN mutants N155H and Q148H without inhibitors demonstrated that their capacity to assemble the synaptic complex and promote concerted integration was similar to their reported virus replication capacities. The concerted integration activity of Q148H showed a higher cross-resistance to raltegravir than observed with N155H, providing evidence as to why the Q148H pathway with secondary mutations is the predominant pathway upon prolonged treatment. Notably, MK-2048 is equally potent against wild-type IN and raltegravir-resistant IN mutant N155H, suggesting this inhibitor may bind similarly within their drug-binding pockets.

Mechanisms of Human Immunodeficiency Virus Type 1 Concerted Integration Related to Strand Transfer Inhibition and Drug Resistance

ABSTRACT The “strand transfer inhibitors” of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3′ OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3′-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.

Different states of avian myeloblastosis virus dna polymerase and their binding capacity to primer tRNATrp

Three different DNA polymerase species can be identified by phosphocellulose chromatography following treatment of the avian myeloblastosis virus αβ DNA polymerase with 1,4-dioxane: (1) α DNA polymerase, (2) residual αβ DNA polymerase, and (3) a species enriched for β DNA polymerase. Binding studies with the major species of primer RNA (tRNATrP) involved in the initiation of RNA-directed DNA synthesis in vitro, were carried out with these three DNA polymerase species. Both αβ and the β DNA polymerase enriched species bound tightly to [32P]tRNATrp as demonstrated by the ability of both enzymes to exclude tRNATrp on Sephadex G-75 columns and by increasing the sedimentation rate of tRNATrp in glycerol gradients. Neither enzyme required divalent metal ion for binding to tRNATrp at 0°. The binding capacity of these two enzymes to tRNATrp was significantly reduced in the presence of MgCl2. This reduction in binding capacity was most likely due to conformational changes of tRNATrp resulting from selective binding with Mg2+. α DNA polymerase, even when present in a 1000-fold molar excess with respect to tRNATrp, did not bind to this primer RNA irrespective of whether or not divalent metal ion was present. These results suggest that the β subunit in αβ DNA polymerase is required for effective binding of the holoenzyme to tRNATrp.

A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3′ DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.

Purification of reverse transcriptase from avian retroviruses using affinity chromatography on heparin-sepharose

Reverse transcriptase from Rous sarcoma virus and avian myeloblastosis virus was purified by a rapid two-step procedure using chromatography on phosphocellulose and heparin-Sepharose. The resulting enzyme was homogeneous, had a high specific activity and was free of contaminating nucleases. This procedure has been adapted to small-scale preparation of enzyme from mutant virus containing thermolabile reverse transcriptase, and is equally suitable for large-scale enzyme purification.