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Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants
Fathima Nuzra Nagoor Pitchai1, Akhil Chameettachal1, Valérie Vivet-Boudou2
1Department of Microbiology & Immunology, College of Medicine and Health Sciences (CMHS), United Arab Emirates University (UAEU), Al Ain, United Arab Emirates.
Abstract:
How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.
Insights
Mason-Pfizer monkey virus Gag protein selects genomic RNA by binding two loops on the packaging signal (Psi). This mechanism distinguishes genomic RNA from spliced viral RNAs, aiding retroviral vector development.
Area of Science:
- * Virology
- * Molecular Biology
- * Gene Therapy
Background:
- * Retroviral virion assembly relies on specific packaging of genomic RNA (gRNA) by Gag proteins.
- * Understanding Gag-gRNA recognition is crucial for retroviral vector safety and efficacy.
- * Mason-Pfizer monkey virus (MPMV) serves as a model to investigate these interactions.
Purpose of the Study:
- * To elucidate the mechanism by which MPMV Gag proteins recognize their packaging signal (Psi) on gRNA.
- * To identify specific binding sites and RNA structures involved in selective gRNA packaging.
- * To inform the design of improved retroviral vectors for gene therapy.
Main Methods:
- * Band-shift assays were employed to study Gag-RNA interactions.
- * Footprinting experiments were utilized to map Gag binding sites on MPMV Psi RNA.
- * MPMV was used as the model retrovirus.
Main Results:
- * MPMV Gag protein (Pr78Gag) binds to two distinct single-stranded loops within the MPMV Psi RNA.
- * These binding sites are a large purine loop (ssPurines) and a GU-rich motif overlapping a purine repeat (bpPurines).
- * The second binding site's location downstream of the major splice donor prevents recognition of spliced viral RNAs.
Conclusions:
- * MPMV Gag protein employs a dual-site RNA recognition mechanism for selective gRNA packaging.
- * This selectivity ensures that only full-length gRNA is packaged, excluding spliced RNAs.
- * Findings contribute to understanding retroviral assembly and developing safer gene therapy vectors.
