Clip for studying protein-RNA interactions that regulate virus replication

Mugisha, Christian Shema; Tenneti, Kasyap; Kutluay, Sebla B.

doi:10.1016/j.ymeth.2019.11.011

Cited by 9 publications

(11 citation statements)

References 45 publications

(54 reference statements)

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“…1D, S1G). Overall, these results demonstrate that the combination of D256N and D270N mutations is sufficient to substantially increase the replication competency of the HIV- virions and visualized per CLIP protocol as described previously (38,72,73). IN-RNA complexes were readily visible for viruses bearing WT IN and the D256N, D270N and D2N substitutions introduced on the WT IN backbone did not impact RNA binding (Fig.…”

Section: Introductionsupporting

confidence: 72%

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

Mugisha

Dinh

Tenneti

et al. 2021

Preprint

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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.AUTHOR SUMMARYIn addition to its well-defined catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome and regulates proper virion maturation. Inhibition of IN binding to the HIV-1 genome through mutations of positively charged residues within the C-terminal domain (CTD, i.e. R269, K273) results in non-infectious particles in which the viral genomes are mislocalized in improperly matured virions. Here we have isolated compensatory mutations in the background of the class II IN (R269A/K273A) mutant virus that restored the ability of IN to bind RNA. We found that additional substitutions of nearby acidic residues (i.e. D256 and D270), which restored the overall positive charge of the CTD, rescued the ability of IN to bind RNA and thus resulted in formation of correctly matured, infectious virions. These compensatory substitutions also revealed the role of specific residues within the CTD that determine sensitivity to allosteric integrase inhibitors (ALLINIs), a class of compounds that indirectly target IN-RNA interactions. Taken together, our findings reveal the importance of the electrostatic interactions between the IN CTD and the gRNA and provide key genetic evidence for a crucial role of the CTD in antiviral activity of ALLINIs.

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Section: Introductionsupporting

confidence: 72%

“…CLIP experiments were conducted as previously described [15,69,70,97,98]. In short, cells in 15 cm cell culture plates were transfected with 30 μg full-length proviral plasmid (pNL4-3) DNA containing the WT sequence or indicated IN mutations.…”

Section: Clip Experimentsmentioning

confidence: 99%

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

Mugisha

Dinh

Tenneti

et al. 2021

Preprint

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“…We next assessed whether the IN D256N and IN D270N substitutions rendered the HIV-1 NL4-3 IN (R269A/K273A) virus replication competent by restoring IN-gRNA binding and proper virion maturation. To this end, IN-gRNA complexes were immunoprecipitated from UV-cross-linked virions and visualized per CLIP protocol as described previously ( 38 , 74 , 75 ). IN-RNA complexes were readily visible for viruses bearing WT IN, and the D256N, D270N, and D2N substitutions introduced on the WT IN backbone did not impact RNA binding ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…CLIP experiments were conducted as previously described ( 15 , 74 , 75 , 98 , 99 ). In short, cells in 15-cm cell culture plates were transfected with 30 μg full-length proviral plasmid (pNL4-3) DNA or derivatives carrying the IN mutations.…”

Section: Methodsmentioning

confidence: 99%

Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA

Mugisha

Dinh

Kumar

et al. 2022

mBio

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“…Other than interacting with positive single-stranded RNA viruses, hnRNPs could also affect life cycle of retrovirus (158). For example, hnRNP A1, AB, H and F were identified as HIV splicing factors that regulate HIV-1 splicing (158,159). The splicing of HIV RNA increases the coding potential of the viral genome and controls viral gene expression.…”

Section: Other Hnrnpsmentioning

confidence: 99%

Multiple functions of heterogeneous nuclear ribonucleoproteins in the positive single-stranded RNA virus life cycle

et al. 2022

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The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses.

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Clip for studying protein-RNA interactions that regulate virus replication

Cited by 9 publications

References 45 publications

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

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

Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA

Multiple functions of heterogeneous nuclear ribonucleoproteins in the positive single-stranded RNA virus life cycle

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