RESUMEN
The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag with His6-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.
RESUMEN
Simian immunodeficiency virus (SIV) is an important lentivirus used as a non-human primate model to study HIV replication, and pathogenesis of human AIDS, as well as a potential vector for human gene therapy. This study investigated the role of single-stranded purines (ssPurines) as potential genomic RNA (gRNA) packaging determinants in SIV replication. Similar ssPurines have been implicated as important motifs for gRNA packaging in many retroviruses like, HIV-1, MPMV, and MMTV by serving as Gag binding sites during virion assembly. In examining the secondary structure of the SIV 5' leader region, as recently deduced using SHAPE methodology, we identified four specific stretches of ssPurines (I-IV) in the region that harbors major packaging determinants of SIV. The significance of these ssPurine motifs were investigated by mutational analysis coupled with a biologically relevant single round of replication assay. These analyses revealed that while ssPurine II was essential, the others (ssPurines I, III, & IV) did not significantly contribute to SIV gRNA packaging. Any mutation in the ssPurine II, such as its deletion or substitution, or other mutations that caused base pairing of ssPurine II loop resulted in near abrogation of RNA packaging, further substantiating the crucial role of ssPurine II and its looped conformation in SIV gRNA packaging. Structure prediction analysis of these mutants further corroborated the biological results and further revealed that the unpaired nature of ssPurine II is critical for its function during SIV RNA packaging perhaps by enabling it to function as a specific binding site for SIV Gag.
Asunto(s)
Regulación Viral de la Expresión Génica , Genoma Viral , Conformación de Ácido Nucleico , Purinas , Virus de la Inmunodeficiencia de los Simios/fisiología , Ensamble de Virus , Animales , Composición de Base , Emparejamiento Base , Genoma Viral/genética , ARN Viral/química , ARN Viral/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Replicación ViralRESUMEN
Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem-loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.
Asunto(s)
Productos del Gen gag/metabolismo , Virus del Tumor Mamario del Ratón/metabolismo , Empalme del ARN , ARN Viral/metabolismo , Ensamble de Virus/genética , Animales , Sitios de Unión/genética , Cartilla de ADN , Dispersión Dinámica de Luz , Productos del Gen gag/genética , Genoma Viral , Virus del Tumor Mamario del Ratón/genética , Ratones , Conformación de Ácido Nucleico , Purinas , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Productos del Gen gag/química , Guanina/química , Virus del Mono Mason-Pfizer/química , ARN Viral/química , Uracilo/química , Animales , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Regulación Viral de la Expresión Génica , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Guanina/metabolismo , Interacciones Huésped-Patógeno , Virus del Mono Mason-Pfizer/genética , Virus del Mono Mason-Pfizer/metabolismo , Conformación de Ácido Nucleico , Papio , Unión Proteica , Conformación Proteica , Huella de Proteína , ARN Viral/genética , ARN Viral/metabolismo , Transducción de Señal , Uracilo/metabolismoRESUMEN
A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified.
RESUMEN
The mouse mammary tumor virus (MMTV) Pr77Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivativesnot the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77Gag-His6-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77Gag-His6-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His6-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77Gag-His6-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77Gag should lay down the foundation towards performing RNAprotein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.