scholarly journals Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

2021 ◽  
Author(s):  
Christian Shema Mugisha ◽  
Tung Dinh ◽  
Kasyap Tenneti ◽  
Jenna Eve Eschbach ◽  
Keanu Davis ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Rna Binding ◽  
Class Ii ◽  
Integrase Inhibitors ◽  
Ribonucleoprotein Complexes ◽  
Compensatory Mutations ◽  
Insight Into ◽  
Hiv 1 ◽  
Molecular Nature

Independent of its catalytic activity, HIV-1 integrase (IN) enzyme regulates proper particle maturation by binding to and packaging the viral RNA genome (gRNA) inside the mature capsid lattice. Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit the binding of IN to the gRNA and cause the formation of non-infectious virions characterized by mislocalization of the viral ribonucleoprotein complexes between the translucent conical capsid lattice and the viral lipid envelope. To gain insight into the molecular nature of IN-gRNA interactions, we have isolated compensatory substitutions in the background of a class II IN (R269A/K273A) variant that directly inhibits IN binding to the gRNA. We found that additional D256N and D270N substitutions in the C-terminal domain (CTD) of IN restored its ability to bind gRNA and led to the formation of infectious particles with correctly matured morphology. Furthermore, reinstating the overall positive electrostatic potential of the CTD through individual D256R or D256K substitutions was sufficient to restore IN-RNA binding and infectivity for the R269A/K273A as well as the R262A/R263A class II IN mutants. The compensatory mutations did not impact functional IN oligomerization, suggesting that they directly contributed to IN binding to the gRNA. Interestingly, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Structural modeling provided further insight into the molecular nature of IN-gRNA interactions and ALLINI mechanism of action. Taken together, our findings highlight an essential role of IN-gRNA interactions for proper virion maturation and reveal the importance of electrostatic interactions between the IN CTD and the gRNA.

scholarly journals Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

2019 ◽  
Author(s):  
Jennifer Elliott ◽  
Jenna E. Eschbach ◽  
Pratibha C. Koneru ◽  
Wen Li ◽  
Maritza Puray Chavez ◽  
...  
Keyword(s):  
Rna Binding ◽  
Critical Role ◽  
Underlying Mechanism ◽  
Class Ii ◽  
Viral Rna ◽  
Target Cells ◽  
Ribonucleoprotein Complexes ◽  
Virion Morphogenesis ◽  
Hiv 1

ABSTRACTA large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

scholarly journals Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jennifer L Elliott ◽  
Jenna E Eschbach ◽  
Pratibha C Koneru ◽  
Wen Li ◽  
Maritza Puray-Chavez ◽  
...  
Keyword(s):  
Rna Binding ◽  
Critical Role ◽  
Underlying Mechanism ◽  
Class Ii ◽  
Viral Rna ◽  
Target Cells ◽  
Ribonucleoprotein Complexes ◽  
Immunodeficiency Virus ◽  
Hiv 1

A large number of human immunodeficiency virus 1 (HIV-1) integrase (IN) alterations, referred to as class II substitutions, exhibit pleiotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of the three distinct mechanisms: (i) markedly reducing IN levels thus precluding the formation of IN complexes with viral RNA; (ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; and (iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in the mislocalization of viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

scholarly journals POLIII-derived non-coding RNAs acting as scaffolds and decoys

2019 ◽  
Vol 11 (10) ◽  
pp. 880-885 ◽  
Author(s):  
Hendrik Täuber ◽  
Stefan Hüttelmaier ◽  
Marcel Köhn
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Current Knowledge ◽  
Cellular Functions ◽  
Control Of Gene Expression ◽  
Ribonucleoprotein Complexes ◽  
Small Ncrnas ◽  
Non Coding Rnas ◽  
And Function

Abstract A large variety of eukaryotic small structured POLIII-derived non-coding RNAs (ncRNAs) have been described in the past. However, for only few, e.g. 7SL and H1/MRP families, cellular functions are well understood. For the vast majority of these transcripts, cellular functions remain unknown. Recent findings on the role of Y RNAs and other POLIII-derived ncRNAs suggest an evolutionarily conserved function of these ncRNAs in the assembly and function of ribonucleoprotein complexes (RNPs). These RNPs provide cellular `machineries’, which are essential for guiding the fate and function of a variety of RNAs. In this review, we summarize current knowledge on the role of POLIII-derived ncRNAs in the assembly and function of RNPs. We propose that these ncRNAs serve as scaffolding factors that `chaperone’ RNA-binding proteins (RBPs) to form functional RNPs. In addition or associated with this role, some small ncRNAs act as molecular decoys impairing the RBP-guided control of RNA fate by competing with other RNA substrates. This suggests that POLIII-derived ncRNAs serve essential and conserved roles in the assembly of larger RNPs and thus the control of gene expression by indirectly guiding the fate of mRNAs and lncRNAs.

scholarly journals Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation

2015 ◽  
Vol 89 (19) ◽  
pp. 9765-9780 ◽  
Author(s):  
Juan Fontana ◽  
Kellie A. Jurado ◽  
Naiqian Cheng ◽  
Ngoc L. Ly ◽  
James R. Fuchs ◽  
...  
Keyword(s):  
Electron Density ◽  
Nucleocapsid Protein ◽  
Class Ii ◽  
Viral Rna ◽  
Wild Type ◽  
Integrase Inhibitors ◽  
Double Stranded Dna ◽  
Condensate Formation ◽  
Internal Electron ◽  
Hiv 1

ABSTRACTDuring virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain “eccentric condensates,” electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from ∼12% to ∼53%). Eccentric condensates have a high NC content, as demonstrated by “tomo-bubblegram” imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN intransas part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation.IMPORTANCEMaturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form “eccentric condensates” outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied intrans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.

scholarly journals The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by a RNA-Mediated Dimerization Mechanism

2017 ◽  
Author(s):  
Nadine M. Shaban ◽  
Ke Shi ◽  
Kate V. Lauer ◽  
Michael A. Carpenter ◽  
Christopher M. Richards ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Binding ◽  
Cytosine Deaminase ◽  
Rnase A ◽  
Binding Motif ◽  
Innate And Adaptive Immunity ◽  
Enzyme Preparations ◽  
Gene Diversification ◽  
Hiv 1

SUMMARYHuman APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA editing enzymes function in innate immunity by restricting the replication of viruses and transposons. Misregulated APOBEC3H also contributes to cancer mutagenesis. Here we address the role of RNA in APOBEC3H regulation. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment yields enzyme preparations with stronger DNA deaminase activity. RNA binding-defective mutants are DNA hypermutators. Chromatography profiles and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. RNA binding is required for APOBEC3H cytoplasmic localization and for packaging into HIV-1 particles and antiviral activity. Related DNA deaminases including other APOBEC3 family members and the antibody gene diversification enzyme AID also bind RNA and are predicted to have a similar RNA binding motif suggesting mechanistic conservation and relevance to innate and adaptive immunity and to multiple diseases.HIGHLIGHTSRNA inhibits human APOBEC3H DNA cytosine deaminase activityRNA binding mutants are DNA hypermutatorsX-ray structure demonstrates an RNA duplex-mediated APOBEC3H dimerization mechanismRNA binding is required for packaging into HIV-1 particles and antiviral activity

scholarly journals An insight into the Inhibition of HIV‐1 replication by co‐infecting HHV‐6, HHV‐7 or GVB‐C viruses: Role of Mutually Homologous miRNAs in Downregulation of Viral Replication

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Omar Bagasra ◽  
Mayank Aggarwal ◽  
krishna chaitanya Addanki ◽  
Mohammad Alsayari ◽  
samina Noorali ◽  
...  
Keyword(s):  
Viral Replication ◽  
Insight Into ◽  
Hiv 1

scholarly journals Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 394 ◽  
Author(s):  
Tomas Kroupa ◽  
Siddhartha A. K. Datta ◽  
Alan Rein
Keyword(s):  
Salt Concentration ◽  
Electrostatic Interactions ◽  
Rna Binding ◽  
High Specificity ◽  
Viral Rna ◽  
Gag Protein ◽  
Gag Polyprotein ◽  
The Individual ◽  
Hiv 1

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag–RNA and Gag–Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.

scholarly journals Circular RNAs: new genetic tools in melanoma

2020 ◽  
Vol 14 (7) ◽  
pp. 563-571 ◽  
Author(s):  
Jamal Hallajzadeh ◽  
Elaheh Amirani ◽  
Hamed Mirzaei ◽  
Rana Shafabakhsh ◽  
Seyyed M Mirhashemi ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Binding ◽  
New Technologies ◽  
Rna Binding Proteins ◽  
Circular Rnas ◽  
Cancer Types ◽  
Microrna Sponge ◽  
The Impact ◽  
Insight Into

Melanoma is the most lethal form of skin cancer. New technologies have resulted in major advances in the diagnosis and treatment of melanoma and other cancer types. Recently, some studies have investigated the role of circular RNAs (circRNAs) in different cancers. CircRNAs are a member of long noncoding RNA family mainly formed through back-splicing and have a closed-loop structure. These molecules affect several biological and oncogenic cascades in diverse ways via acting as microRNA sponge, interacting with RNA-binding proteins and acting as a transcription regulator. In this review, we made an insight into the impact of circRNA dysregulation in the melanoma tumorigenesis based on the presented evidences.

scholarly journals MicroED structures of HIV-1 Gag CTD-SP1 reveal binding interactions with the maturation inhibitor bevirimat

2018 ◽  
Vol 115 (52) ◽  
pp. 13258-13263 ◽  
Author(s):  
Michael D. Purdy ◽  
Dan Shi ◽  
Jakub Chrustowicz ◽  
Johan Hattne ◽  
Tamir Gonen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Binding Interactions ◽  
Structure Based Drug Design ◽  
Gag Polyprotein ◽  
Pentacyclic Triterpenoid ◽  
Maturation Inhibitor ◽  
Insight Into ◽  
Hiv 1

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

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