Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA
ABSTRACT HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the cap...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-10-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.00431-22 |
| _version_ | 1828729841401200640 |
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| author | Christian Shema Mugisha Tung Dinh Abhishek Kumar Kasyap Tenneti Jenna E. Eschbach Keanu Davis Robert Gifford Mamuka Kvaratskhelia Sebla B. Kutluay |
| author_facet | Christian Shema Mugisha Tung Dinh Abhishek Kumar Kasyap Tenneti Jenna E. Eschbach Keanu Davis Robert Gifford Mamuka Kvaratskhelia Sebla B. Kutluay |
| author_sort | Christian Shema Mugisha |
| collection | DOAJ |
| description | ABSTRACT HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA. IMPORTANCE In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues (i.e., R262, R263, R269, K273) within its C-terminal domain (CTD) and regulates proper virion maturation. Mutation of these residues results in the formation of morphologically aberrant viruses blocked at an early reverse transcription stage in cells. Here we show that compensatory substitutions in nearby negatively charged aspartic acid residues (i.e., D256N, D270N) restore the ability of IN to bind gRNA for these mutant viruses and result in the formation of accurately matured infectious virions. Similarly, individual charge reversal substitutions at D256 as well as other nearby positions (i.e., D278, D279) are all sufficient to enable the respective IN mutants to bind gRNA, and subsequently restore reverse transcription and virion infectivity. Taken together, our findings reveal the importance of highly pliable electrostatic interactions in IN-gRNA binding. |
| first_indexed | 2024-04-12T14:38:41Z |
| format | Article |
| id | doaj.art-3805d4366b47469996c781fa72fa501d |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-12T14:38:41Z |
| publishDate | 2022-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-3805d4366b47469996c781fa72fa501d2022-12-22T03:28:58ZengAmerican Society for MicrobiologymBio2150-75112022-10-0113510.1128/mbio.00431-22Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNAChristian Shema Mugisha0Tung Dinh1Abhishek Kumar2Kasyap Tenneti3Jenna E. Eschbach4Keanu Davis5Robert Gifford6Mamuka Kvaratskhelia7Sebla B. Kutluay8Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USADivision of Infectious Diseases, University of Colorado School of Medicine, Aurora, Colorado, USADepartment of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USADepartment of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USADepartment of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USADepartment of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USAMRC-University of Glasgow Centre for Virus Research, Bearsden, Glasgow, United KingdomDivision of Infectious Diseases, University of Colorado School of Medicine, Aurora, Colorado, USADepartment of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, USAABSTRACT HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA. IMPORTANCE In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues (i.e., R262, R263, R269, K273) within its C-terminal domain (CTD) and regulates proper virion maturation. Mutation of these residues results in the formation of morphologically aberrant viruses blocked at an early reverse transcription stage in cells. Here we show that compensatory substitutions in nearby negatively charged aspartic acid residues (i.e., D256N, D270N) restore the ability of IN to bind gRNA for these mutant viruses and result in the formation of accurately matured infectious virions. Similarly, individual charge reversal substitutions at D256 as well as other nearby positions (i.e., D278, D279) are all sufficient to enable the respective IN mutants to bind gRNA, and subsequently restore reverse transcription and virion infectivity. Taken together, our findings reveal the importance of highly pliable electrostatic interactions in IN-gRNA binding.https://journals.asm.org/doi/10.1128/mbio.00431-22HIV-1integraseprotein-RNA interactionsvirion maturationvirology |
| spellingShingle | Christian Shema Mugisha Tung Dinh Abhishek Kumar Kasyap Tenneti Jenna E. Eschbach Keanu Davis Robert Gifford Mamuka Kvaratskhelia Sebla B. Kutluay Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA mBio HIV-1 integrase protein-RNA interactions virion maturation virology |
| title | Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA |
| title_full | Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA |
| title_fullStr | Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA |
| title_full_unstemmed | Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA |
| title_short | Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA |
| title_sort | emergence of compensatory mutations reveals the importance of electrostatic interactions between hiv 1 integrase and genomic rna |
| topic | HIV-1 integrase protein-RNA interactions virion maturation virology |
| url | https://journals.asm.org/doi/10.1128/mbio.00431-22 |
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