Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

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Among retroviral integrases prototype foamy virus integrase displays unique biochemical activities

10.1101/2021.11.17.469013 ◽

2021 ◽

Author(s):

Anthony J Rabe ◽

Yow Yong Tan ◽

Ross C Larue ◽

Kristine E Yoder

Keyword(s):

Foamy Virus ◽

Inverse Correlation ◽

Divalent Metal ◽

Viral Dna ◽

Prototype Foamy Virus ◽

Dna Oligonucleotides ◽

Low Concentrations ◽

Target Dna ◽

One Step ◽

Hiv 1

Integrase enzymes of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 seconds, PFV intasomes do not commit to target DNA during their reaction lifetime. Together these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

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

Journal of Virology ◽

10.1128/jvi.02863-06 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12189-12199 ◽

Cited By ~ 32

Author(s):

Krishan K. Pandey ◽

Sibes Bera ◽

Jacob Zahm ◽

Ajaykumar Vora ◽

Kara Stillmock ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Dependent Manner ◽

Strand Transfer ◽

Viral Dna ◽

Target Dna ◽

Immunodeficiency Virus ◽

Dna Ends ◽

Hiv 1

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.

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Molecular Dynamics Approaches Estimate the Binding Energy of HIV-1 Integrase Inhibitors and Correlate withIn VitroActivity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05292-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 411-419 ◽

Cited By ~ 30

Author(s):

Barry C. Johnson ◽

Mathieu Métifiot ◽

Yves Pommier ◽

Stephen H. Hughes

Keyword(s):

Molecular Dynamics ◽

Binding Energy ◽

Foamy Virus ◽

Complex Model ◽

Integrase Inhibitors ◽

Training Set ◽

Viral Dna ◽

Starting Point ◽

Hiv 1

ABSTRACTThe design of novel integrase (IN) inhibitors has been aided by recent crystal structures revealing the binding mode of these compounds with a full-length prototype foamy virus (PFV) IN and synthetic viral DNA ends. Earlier docking studies relied on incomplete structures and did not include the contribution of the viral DNA to inhibitor binding. Using the structure of PFV IN as the starting point, we generated a model of the corresponding HIV-1 complex and developed a molecular dynamics (MD)-based approach that correlates with thein vitroactivities of novel compounds. Four well-characterized compounds (raltegravir, elvitegravir, MK-0536, and dolutegravir) were used as a training set, and the data for theirin vitroactivity against the Y143R, N155H, and G140S/Q148H mutants were used in addition to the wild-type (WT) IN data. Three additional compounds were docked into the IN-DNA complex model and subjected to MD simulations. All three gave interaction potentials within 1 standard deviation of values estimated from the training set, and the most active compound was identified. Additional MD analysis of the raltegravir- and dolutegravir-bound complexes gave internal and interaction energy values that closely match the experimental binding energy of a compound related to raltegravir that has similar activity. These approaches can be used to gain a deeper understanding of the interactions of the inhibitors with the HIV-1 intasome and to identify promising scaffolds for novel integrase inhibitors, in particular, compounds that retain activity against a range of drug-resistant mutants, making it possible to streamline synthesis and testing.

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Short Communication: Efficiently Inhibiting HIV-1 Replication by a Prototype Foamy Virus Vector Expressing Novel H1 Promoter-Driven Short Hairpin RNAs

AIDS Research and Human Retroviruses ◽

10.1089/aid.2014.0217 ◽

2015 ◽

Vol 31 (2) ◽

pp. 183-188 ◽

Cited By ~ 3

Author(s):

Qingqing Cheng ◽

Lanlan Dong ◽

Fengfeng Zhang ◽

Peipei Yuan ◽

Zhi Li ◽

...

Keyword(s):

Short Communication ◽

Foamy Virus ◽

Virus Vector ◽

Prototype Foamy Virus ◽

Short Hairpin ◽

Short Hairpin Rnas ◽

Hiv 1

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Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

10.1101/2020.03.17.994137 ◽

2020 ◽

Cited By ~ 3

Author(s):

Kenneth N. Hass ◽

Mengdi Bao ◽

Qian He ◽

Myeongkee Park ◽

Peiwu Qin ◽

...

Keyword(s):

Point Of Care ◽

Molecular Diagnostic ◽

Nucleic Acid Detection ◽

Viral Dna ◽

Fully Integrated ◽

Impact System ◽

Target Dna ◽

Reporter Probe ◽

One Step ◽

Labeled Probe

ABSTRACTA fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed at isothermal conditions (37°C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.

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Efficient Concerted Integration by Recombinant Human Immunodeficiency Virus Type 1 Integrase without Cellular or Viral Cofactors

Journal of Virology ◽

10.1128/jvi.76.7.3105-3113.2002 ◽

2002 ◽

Vol 76 (7) ◽

pp. 3105-3113 ◽

Cited By ~ 56

Author(s):

Sapna Sinha ◽

Michael H. Pursley ◽

Duane P. Grandgenett

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Type I ◽

Wild Type ◽

Viral Dna ◽

Target Dna ◽

Immunodeficiency Virus ◽

Nucleoprotein Complexes ◽

Hiv 1

ABSTRACT Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At ∼30 nM IN (20 min at 37°C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of ∼70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (∼125 μg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.

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