Our Technophile
Dr. Lizna M Ali
Medical Research Specialist
Dr. Ali is the seniormost member of the laboratory and her research interests are founded in retroviral RNA packaging (Ali et al., Viruses 2016). She has recently delineated the role of purine-rich regions in MPMV genomic RNA packaging (Ali et al., Front. Micro., 2020). She is currently working on MMTV gRNA export pathway. Briefly, MMTV encodes an RNA export protein called Rem, which interacts with the Rem-Responsive Element (RmRE) present at the 3’end of the viral genome, facilitating nuclear export of unspliced RNA. Dr. Ali is working towards identifying the Rem binding site(s) on RmRE by expressing and purifying Rem protein from bacterial cultures.
Dr. Ali obtained her MS degree in Biomedical Genetics from the Vellore Institute of Technology (2005), India, followed by her PhD degree from University of Pecs, Hungary in 2021.
E-mail: [email protected]
Publications
2026
Jehad, Mohammad Abdullah; Ali, Lizna M; Pillai, Vineeta N; Prabhu, Suresha G; Mustafa, Farah; Rizvi, Tahir A
Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication Journal Article
In: FEMS Microbiology Reviews, vol. 50, 2026, ISSN: 1574-6976.
@article{Jehad2025b,
title = {Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication},
author = {Mohammad Abdullah Jehad and Lizna M Ali and Vineeta N Pillai and Suresha G Prabhu and Farah Mustafa and Tahir A Rizvi},
editor = {Christian Münz},
doi = {10.1093/femsre/fuaf066},
issn = {1574-6976},
year = {2026},
date = {2026-01-10},
urldate = {2026-01-10},
journal = {FEMS Microbiology Reviews},
volume = {50},
publisher = {Oxford University Press (OUP)},
abstract = {Retroviruses are exclusive group of positive-sense RNA viruses defined by their ability to reverse transcribe their RNA genome and integrate it into the host’s chromosomal DNA. This distinctive replication strategy enables persistent infection and has profoundly shaped our understanding of molecular biology, gene regulation, and evolution. Retroviruses have contributed to landmark discoveries, including the identification of oncogenes, mechanisms of transcriptional control, and the development of gene therapy vectors. This review provides an updated overview of retroviral molecular biology, emphasizing the coordinated steps of the viral life cycle and emerging insights that are reshaping classical models. It explores virion structure, genome organization, and the interplay of cis-acting sequences and trans-acting factors that govern replication. Special focus is given to recent advances in understanding nuclear trafficking of capsids, spatial dynamics of reverse transcription and integration leading to provirus formation, RNA nuclear export, and selective genome packaging. The structural and functional roles of viral proteins, particularly Gag, are discussed in the context of assembly and maturation. By integrating foundational concepts with new discoveries, this review highlights the molecular sophistication of retroviral replication and identifies outstanding questions that guide future research, with implications extending to antiviral strategies, gene therapy, cancer biology, and evolution.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Retroviruses are exclusive group of positive-sense RNA viruses defined by their ability to reverse transcribe their RNA genome and integrate it into the host’s chromosomal DNA. This distinctive replication strategy enables persistent infection and has profoundly shaped our understanding of molecular biology, gene regulation, and evolution. Retroviruses have contributed to landmark discoveries, including the identification of oncogenes, mechanisms of transcriptional control, and the development of gene therapy vectors. This review provides an updated overview of retroviral molecular biology, emphasizing the coordinated steps of the viral life cycle and emerging insights that are reshaping classical models. It explores virion structure, genome organization, and the interplay of cis-acting sequences and trans-acting factors that govern replication. Special focus is given to recent advances in understanding nuclear trafficking of capsids, spatial dynamics of reverse transcription and integration leading to provirus formation, RNA nuclear export, and selective genome packaging. The structural and functional roles of viral proteins, particularly Gag, are discussed in the context of assembly and maturation. By integrating foundational concepts with new discoveries, this review highlights the molecular sophistication of retroviral replication and identifies outstanding questions that guide future research, with implications extending to antiviral strategies, gene therapy, cancer biology, and evolution.
2025
Jaballah, Soumeya Ali; Ali, Lizna M; Jehad, Mohammad Abdullah; Akhlaq, Shaima; Rizvi, Tahir A.; Mustafa, Farah
Retroviral Vector Technology for Gene Therapy: History, Current Landscape, and Future Prospects Journal Article
In: Journal of Molecular Biology, vol. 437, no. 24, 2025, ISSN: 0022-2836.
@article{Jaballah2025,
title = {Retroviral Vector Technology for Gene Therapy: History, Current Landscape, and Future Prospects},
author = {Soumeya Ali Jaballah and Lizna M Ali and Mohammad Abdullah Jehad and Shaima Akhlaq and Tahir A. Rizvi and Farah Mustafa},
doi = {10.1016/j.jmb.2025.169473},
issn = {0022-2836},
year = {2025},
date = {2025-12-00},
urldate = {2025-12-00},
journal = {Journal of Molecular Biology},
volume = {437},
number = {24},
publisher = {Elsevier BV},
abstract = {The concept of gene therapy and its practice has been prevalent for over five decades. The first successful retroviral vector-based gene therapy trial took place ∼35 years ago, followed by several setbacks. However, recent years have seen a surge in successes, offering new hope to patients with genetic and other disorders once deemed untreatable. Over the past decade, rapid advancements in molecular biology have led to the development of safer and more effective gene therapy strategies with various gene delivery systems now in use. Among these, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are the most widely employed in both research and clinical settings. This is due to their natural efficiency in delivering genetic material into target cells. Among these viral vectors, retroviruses stand out for their unique ability to reverse-transcribe and integrate their genetic material into the host genome, ensuring stable and long-term gene expression. This review highlights advances in retroviral vector development, examining both their therapeutic potential and associated challenges. It also explores strategies for vector production, including transient and stable systems tailored to meet clinical and regulatory demands. Significant progress is discussed in mitigating insertional mutagenesis and vector silencing. As a result, next-generation retroviral vectors with improved safety and efficacy have made it past regulatory approval and are commercially available. Current innovations include replication-competent, non-integrating, integration-retargeted, and hybrid CRISPR/Cas-expressing retroviral vectors undergoing pre-clinical and clinical investigations. This reflects a new era in gene therapy, with retroviral vectors reimagined for greater precision, control, and therapeutic impact.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The concept of gene therapy and its practice has been prevalent for over five decades. The first successful retroviral vector-based gene therapy trial took place ∼35 years ago, followed by several setbacks. However, recent years have seen a surge in successes, offering new hope to patients with genetic and other disorders once deemed untreatable. Over the past decade, rapid advancements in molecular biology have led to the development of safer and more effective gene therapy strategies with various gene delivery systems now in use. Among these, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are the most widely employed in both research and clinical settings. This is due to their natural efficiency in delivering genetic material into target cells. Among these viral vectors, retroviruses stand out for their unique ability to reverse-transcribe and integrate their genetic material into the host genome, ensuring stable and long-term gene expression. This review highlights advances in retroviral vector development, examining both their therapeutic potential and associated challenges. It also explores strategies for vector production, including transient and stable systems tailored to meet clinical and regulatory demands. Significant progress is discussed in mitigating insertional mutagenesis and vector silencing. As a result, next-generation retroviral vectors with improved safety and efficacy have made it past regulatory approval and are commercially available. Current innovations include replication-competent, non-integrating, integration-retargeted, and hybrid CRISPR/Cas-expressing retroviral vectors undergoing pre-clinical and clinical investigations. This reflects a new era in gene therapy, with retroviral vectors reimagined for greater precision, control, and therapeutic impact.
2024
Prabhu, Suresha G.; Pillai, Vineeta N.; Ali, Lizna Mohamed; Vivet-Boudou, Valérie; Chameettachal, Akhil; Bernacchi, Serena; Mustafa, Farah; Marquet, Roland; Rizvi, T. A.
MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals Journal Article
In: PLOS Biology, vol. 22, no. 10, pp. 1-34, 2024.
@article{10.1371/journal.pbio.3002827,
title = {MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals},
author = {Suresha G. Prabhu and Vineeta N. Pillai and Lizna Mohamed Ali and Valérie Vivet-Boudou and Akhil Chameettachal and Serena Bernacchi and Farah Mustafa and Roland Marquet and T. A. Rizvi},
url = {https://doi.org/10.1371/journal.pbio.3002827},
doi = {10.1371/journal.pbio.3002827},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {PLOS Biology},
volume = {22},
number = {10},
pages = {1-34},
publisher = {Public Library of Science},
abstract = {The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type–like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type–like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently.
Krishnan, A.; Ali, L. M.; Prabhu, Suresha G; Pillai, V. N.; Chameettachal, A.; Vivet-Boudou, Valérie; Bernacchi, Serena; Mustafa, F.; Marquet, Roland; Rizvi, T. A.
Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging Journal Article
In: RNA, vol. 30, no. 1, pp. 68–88, 2024, ISSN: 1469-9001.
@article{Krishnan2023,
title = {Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging},
author = {A. Krishnan and L. M. Ali and Suresha G Prabhu and V. N. Pillai and A. Chameettachal and Valérie Vivet-Boudou and Serena Bernacchi and F. Mustafa and Roland Marquet and T. A. Rizvi},
doi = {10.1261/rna.079840.123},
issn = {1469-9001},
year = {2024},
date = {2024-01-00},
urldate = {2024-01-00},
journal = {RNA},
volume = {30},
number = {1},
pages = {68--88},
publisher = {Cold Spring Harbor Laboratory},
abstract = {The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88 stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gag on WT and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA–Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.
2023
Pillai, V. N.; Ali, L. M.; Prabhu, S. G.; Krishnan, A.; Tariq, S.; Mustafa, F.; Rizvi, T. A.
Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55(Gag) Journal Article
In: Heliyon, vol. 9, no. 1, pp. e12892, 2023, ISSN: 2405-8440 (Print) 2405-8440.
@article{RN115,
title = {Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency virus (SIV) Pr55(Gag)},
author = {V. N. Pillai and L. M. Ali and S. G. Prabhu and A. Krishnan and S. Tariq and F. Mustafa and T. A. Rizvi},
doi = {10.1016/j.heliyon.2023.e12892},
issn = {2405-8440 (Print) 2405-8440},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Heliyon},
volume = {9},
number = {1},
pages = {e12892},
abstract = {The simian immunodeficiency virus (SIV) precursor polypeptide Pr55(Gag) 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 Pr55(Gag) by expressing its different components independently, studies using full-length SIV Pr55(Gag) have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55(Gag). We successfully expressed soluble, full-length SIV Pr55(Gag) with His(6)-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 Pr55(Gag) which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The simian immunodeficiency virus (SIV) precursor polypeptide Pr55(Gag) 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 Pr55(Gag) by expressing its different components independently, studies using full-length SIV Pr55(Gag) have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55(Gag). We successfully expressed soluble, full-length SIV Pr55(Gag) with His(6)-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 Pr55(Gag) which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.
2021
Chameettachal, A.; Vivet-Boudou, V.; Pitchai, F. N. N.; Pillai, V. N.; Ali, L. M.; Krishnan, A.; Bernacchi, S.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag Journal Article
In: Nucleic Acids Res, vol. 49, no. 8, pp. 4668-4688, 2021, ISSN: 0305-1048 (Print) 0305-1048.
@article{RN40,
title = {A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag},
author = {A. Chameettachal and V. Vivet-Boudou and F. N. N. Pitchai and V. N. Pillai and L. M. Ali and A. Krishnan and S. Bernacchi and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.1093/nar/gkab223},
issn = {0305-1048 (Print) 0305-1048},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Nucleic Acids Res},
volume = {49},
number = {8},
pages = {4668-4688},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
Pillai, V. N.; Ali, L. M.; Prabhu, S. G.; Krishnan, A.; Chameettachal, A.; Pitchai, F. N. N.; Mustafa, F.; Rizvi, T. A.
A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions Journal Article
In: J Mol Biol, vol. 433, no. 23, pp. 167293, 2021, ISSN: 0022-2836.
@article{RN38,
title = {A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions},
author = {V. N. Pillai and L. M. Ali and S. G. Prabhu and A. Krishnan and A. Chameettachal and F. N. N. Pitchai and F. Mustafa and T. A. Rizvi},
doi = {10.1016/j.jmb.2021.167293},
issn = {0022-2836},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {J Mol Biol},
volume = {433},
number = {23},
pages = {167293},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
Pitchai, F. N. N.; Chameettachal, A.; Vivet-Boudou, V.; Ali, L. M.; Pillai, V. N.; Krishnan, A.; Bernacchi, S.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
Identification of Pr78(Gag) Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants Journal Article
In: J Mol Biol, vol. 433, no. 10, pp. 166923, 2021, ISSN: 0022-2836.
@article{RN19,
title = {Identification of Pr78(Gag) Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants},
author = {F. N. N. Pitchai and A. Chameettachal and V. Vivet-Boudou and L. M. Ali and V. N. Pillai and A. Krishnan and S. Bernacchi and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.1016/j.jmb.2021.166923},
issn = {0022-2836},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {J Mol Biol},
volume = {433},
number = {10},
pages = {166923},
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 Pr78(Gag) 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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 Pr78(Gag) 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.
2020
Ali, L. M.; Pitchai, F. N. N.; Vivet-Boudou, V.; Chameettachal, A.; Jabeen, A.; Pillai, V. N.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation Journal Article
In: Front Microbiol, vol. 11, pp. 595410, 2020, ISSN: 1664-302X (Print) 1664-302x.
@article{RN36,
title = {Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation},
author = {L. M. Ali and F. N. N. Pitchai and V. Vivet-Boudou and A. Chameettachal and A. Jabeen and V. N. Pillai and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.3389/fmicb.2020.595410},
issn = {1664-302X (Print) 1664-302x},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Front Microbiol},
volume = {11},
pages = {595410},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
2019
Krishnan, A.; Pillai, Vineeta N.; Chameettachal, Akhil; Ali, Lizna Mohamed; Pitchai, Fathima Nuzra Nagoor; Tariq, Saeed; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.
Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag Journal Article
In: Viruses, vol. 11, no. 8, 2019, ISSN: 1999-4915.
@article{Krishnan2019,
title = {Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag},
author = {A. Krishnan and Vineeta N. Pillai and Akhil Chameettachal and Lizna Mohamed Ali and Fathima Nuzra Nagoor Pitchai and Saeed Tariq and Farah Mustafa and Roland Marquet and Tahir A. Rizvi},
doi = {10.3390/v11080689},
issn = {1999-4915},
year = {2019},
date = {2019-08-00},
urldate = {2019-08-00},
journal = {Viruses},
volume = {11},
number = {8},
publisher = {MDPI AG},
abstract = {<jats:p>The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.</jats:p>},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
<jats:p>The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.</jats:p>
Kalloush, R. M.; Vivet-Boudou, V.; Ali, L. M.; Pillai, V. N.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
In: RNA Biol, vol. 16, no. 5, pp. 612-625, 2019, ISSN: 1547-6286 (Print) 1547-6286.
@article{RN53,
title = {Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging},
author = {R. M. Kalloush and V. Vivet-Boudou and L. M. Ali and V. N. Pillai and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.1080/15476286.2019.1572424},
issn = {1547-6286 (Print) 1547-6286},
year = {2019},
date = {2019-01-01},
urldate = {2019-01-01},
journal = {RNA Biol},
volume = {16},
number = {5},
pages = {612-625},
abstract = {The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging signal is a highly-structured element with several stem-loops held together by two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences. These LRIs play a critical role in maintaining the structure of the 5´ end of the MPMV gRNA. Thus, one could hypothesize that the overall RNA secondary structure of this region is further architecturally held together by three other stem loops (SL3, Gag SL1, and Gag SL2) comprising of sequences from the distal parts of the 5´untranslated region (5' UTR) to ~ 120 nucleotides into gag, excluding gag sequences involved in forming the U5-Gag LRIs. To provide functional evidence for the biological significance of these stem loops during gRNA encapsidation, these structural motifs were mutated and their effects on MPMV RNA packaging and propagation were tested in a single round trans-complementation assay. The mutant RNA structures were further studied by high throughput SHAPE (hSHAPE) assay. Our results reveal that sequences involved in forming these three stem loops do not play crucial roles at an individual level during MPMV gRNA packaging or propagation. Further structure-function analysis indicates that the U5-Gag LRIs have a more important architectural role in stabilizing the higher order structure of the 5´ UTR than the three stem loops which have a more secondary and perhaps indirect role in stabilizing the overall RNA secondary structure of the region. Our work provides a better understanding of the molecular interactions that take place during MPMV gRNA packaging.
2018
Akhlaq, S.; Panicker, N. G.; Philip, P. S.; Ali, L. M.; Dudley, J. P.; Rizvi, T. A.; Mustafa, F.
A cis-Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability Journal Article
In: J Mol Biol, vol. 430, no. 21, pp. 4307-4324, 2018, ISSN: 0022-2836 (Print) 0022-2836.
@article{RN32,
title = {A cis-Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability},
author = {S. Akhlaq and N. G. Panicker and P. S. Philip and L. M. Ali and J. P. Dudley and T. A. Rizvi and F. Mustafa},
doi = {10.1016/j.jmb.2018.08.025},
issn = {0022-2836 (Print) 0022-2836},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {J Mol Biol},
volume = {430},
number = {21},
pages = {4307-4324},
abstract = {BACKGROUND: The mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3' cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5' end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3' RmRE could facilitate translation of all other mRNAs, including gRNA. RESULTS: To address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. CONCLUSIONS: MMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: The mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3' cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5' end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3' RmRE could facilitate translation of all other mRNAs, including gRNA. RESULTS: To address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. CONCLUSIONS: MMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.
Chameettachal, A.; Pillai, V. N.; Ali, L. M.; Pitchai, F. N. N.; Ardah, M. T.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77(Gag) Expressed in Prokaryotic and Eukaryotic Cells Journal Article
In: Viruses, vol. 10, no. 6, 2018, ISSN: 1999-4915.
@article{RN88,
title = {Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77(Gag) Expressed in Prokaryotic and Eukaryotic Cells},
author = {A. Chameettachal and V. N. Pillai and L. M. Ali and F. N. N. Pitchai and M. T. Ardah and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.3390/v10060334},
issn = {1999-4915},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Viruses},
volume = {10},
number = {6},
abstract = {The mouse mammary tumor virus (MMTV) Pr77(Gag) 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 derivatives—not 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 Pr77(Gag)-His₆-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77(Gag)-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 Pr77(Gag)-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 Pr77(Gag) should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The mouse mammary tumor virus (MMTV) Pr77(Gag) 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 derivatives—not 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 Pr77(Gag)-His₆-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77(Gag)-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 Pr77(Gag)-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 Pr77(Gag) should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.
Mustafa, F.; Vivet-Boudou, V.; Jabeen, A.; Ali, L. M.; Kalloush, R. M.; Marquet, R.; Rizvi, T. A.
The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA Journal Article
In: RNA Biol, vol. 15, no. 8, pp. 1047-1059, 2018, ISSN: 1547-6286 (Print) 1547-6286.
@article{RN90,
title = {The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA},
author = {F. Mustafa and V. Vivet-Boudou and A. Jabeen and L. M. Ali and R. M. Kalloush and R. Marquet and T. A. Rizvi},
doi = {10.1080/15476286.2018.1486661},
issn = {1547-6286 (Print) 1547-6286},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {RNA Biol},
volume = {15},
number = {8},
pages = {1047-1059},
abstract = {Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.
Pitchai, F. N. N.; Ali, L.; Pillai, V. N.; Chameettachal, A.; Ashraf, S. S.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
Expression, purification, and characterization of biologically active full-length Mason-Pfizer monkey virus (MPMV) Pr78(Gag) Journal Article
In: Sci Rep, vol. 8, no. 1, pp. 11793, 2018, ISSN: 2045-2322.
@article{RN51,
title = {Expression, purification, and characterization of biologically active full-length Mason-Pfizer monkey virus (MPMV) Pr78(Gag)},
author = {F. N. N. Pitchai and L. Ali and V. N. Pillai and A. Chameettachal and S. S. Ashraf and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.1038/s41598-018-30142-0},
issn = {2045-2322},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Sci Rep},
volume = {8},
number = {1},
pages = {11793},
abstract = {MPMV precursor polypeptide Pr78(Gag) orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78(Gag) either with or without His(6)-tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78(Gag) protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78(Gag) with or without His(6)-tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His(6)-tag to the full-length Pr78(Gag) did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78(Gag), which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
MPMV precursor polypeptide Pr78(Gag) orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78(Gag) either with or without His(6)-tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78(Gag) protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78(Gag) with or without His(6)-tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His(6)-tag to the full-length Pr78(Gag) did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78(Gag), which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.
2016
Ali, L. M.; Rizvi, T. A.; Mustafa, F.
Cross- and Co-Packaging of Retroviral RNAs and Their Consequences Journal Article
In: Viruses, vol. 8, no. 10, 2016, ISSN: 1999-4915.
@article{RN15,
title = {Cross- and Co-Packaging of Retroviral RNAs and Their Consequences},
author = {L. M. Ali and T. A. Rizvi and F. Mustafa},
doi = {10.3390/v8100276},
issn = {1999-4915},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Viruses},
volume = {8},
number = {10},
abstract = {Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP) particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA) among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP) particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA) among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy.
Kalloush, R. M.; Vivet-Boudou, V.; Ali, L. M.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences Journal Article
In: Rna, vol. 22, no. 6, pp. 905-19, 2016, ISSN: 1355-8382 (Print) 1355-8382.
@article{RN77,
title = {Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences},
author = {R. M. Kalloush and V. Vivet-Boudou and L. M. Ali and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.1261/rna.055731.115},
issn = {1355-8382 (Print) 1355-8382},
year = {2016},
date = {2016-01-01},
urldate = {2016-01-01},
journal = {Rna},
volume = {22},
number = {6},
pages = {905-19},
abstract = {MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses.
2015
Ahmad, M. Al; Mustafa, F.; Ali, L. M.; Karakkat, J. V.; Rizvi, T. A.
Label-free capacitance-based identification of viruses Journal Article
In: Sci Rep, vol. 5, pp. 9809, 2015, ISSN: 2045-2322.
@article{RN21,
title = {Label-free capacitance-based identification of viruses},
author = {M. Al Ahmad and F. Mustafa and L. M. Ali and J. V. Karakkat and T. A. Rizvi},
doi = {10.1038/srep09809},
issn = {2045-2322},
year = {2015},
date = {2015-01-01},
urldate = {2015-01-01},
journal = {Sci Rep},
volume = {5},
pages = {9809},
abstract = {This study was undertaken to quantitate a single virus suspension in culture medium without any pre-processing. The electrical capacitance per virus particle was used to identify the kind of virus present by measuring the suspension (virus plus medium) capacitance, de-embedding the medium contribution, and dividing by the virus count. The proposed technique is based on finding the single virus effective dielectric constant which is directly related to the virus composition. This value was used to identify the virus type accordingly. Two types of viruses thus tested were further quantified by a biochemical technique to validate the results. Furthermore, non-organic nanoparticles with known concentration and capacitance per particle were identified using the proposed method. The selectivity of the method was demonstrated by performing electrical measurements on a third virus, revealing that the proposed technique is specific and sensitive enough to permit detection of a few hundred virus particles per milliliter within a few minutes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
This study was undertaken to quantitate a single virus suspension in culture medium without any pre-processing. The electrical capacitance per virus particle was used to identify the kind of virus present by measuring the suspension (virus plus medium) capacitance, de-embedding the medium contribution, and dividing by the virus count. The proposed technique is based on finding the single virus effective dielectric constant which is directly related to the virus composition. This value was used to identify the virus type accordingly. Two types of viruses thus tested were further quantified by a biochemical technique to validate the results. Furthermore, non-organic nanoparticles with known concentration and capacitance per particle were identified using the proposed method. The selectivity of the method was demonstrated by performing electrical measurements on a third virus, revealing that the proposed technique is specific and sensitive enough to permit detection of a few hundred virus particles per milliliter within a few minutes.
2014
Aktar, S. J.; Vivet-Boudou, V.; Ali, L. M.; Jabeen, A.; Kalloush, R. M.; Richer, D.; Mustafa, F.; Marquet, R.; Rizvi, T. A.
Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV) Journal Article
In: Retrovirology, vol. 11, pp. 96, 2014, ISSN: 1742-4690.
@article{RN59,
title = {Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV)},
author = {S. J. Aktar and V. Vivet-Boudou and L. M. Ali and A. Jabeen and R. M. Kalloush and D. Richer and F. Mustafa and R. Marquet and T. A. Rizvi},
doi = {10.1186/s12977-014-0096-6},
issn = {1742-4690},
year = {2014},
date = {2014-01-01},
urldate = {2014-01-01},
journal = {Retrovirology},
volume = {11},
pages = {96},
abstract = {BACKGROUND: One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5' end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5' untranslated region (5' UTR) and the beginning of gag. RESULTS: The RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2'hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5' region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5' CGGCCG 3') at the 5' UTR and contains a canonical "GC" dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging. CONCLUSIONS: Employing structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5' end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5' untranslated region (5' UTR) and the beginning of gag. RESULTS: The RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2'hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5' region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5' CGGCCG 3') at the 5' UTR and contains a canonical "GC" dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging. CONCLUSIONS: Employing structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle.
Ahmad, M. Al; Mustafa, F.; Ali, L. M.; Rizvi, T. A.
Virus detection and quantification using electrical parameters Journal Article
In: Sci Rep, vol. 4, pp. 6831, 2014, ISSN: 2045-2322.
@article{RN24,
title = {Virus detection and quantification using electrical parameters},
author = {M. Al Ahmad and F. Mustafa and L. M. Ali and T. A. Rizvi},
doi = {10.1038/srep06831},
issn = {2045-2322},
year = {2014},
date = {2014-01-01},
urldate = {2014-01-01},
journal = {Sci Rep},
volume = {4},
pages = {6831},
abstract = {Here we identify and quantitate two similar viruses, human and feline immunodeficiency viruses (HIV and FIV), suspended in a liquid medium without labeling, using a semiconductor technique. The virus count was estimated by calculating the impurities inside a defined volume by observing the change in electrical parameters. Empirically, the virus count was similar to the absolute value of the ratio of the change of the virus suspension dopant concentration relative to the mock dopant over the change in virus suspension Debye volume relative to mock Debye volume. The virus type was identified by constructing a concentration-mobility relationship which is unique for each kind of virus, allowing for a fast (within minutes) and label-free virus quantification and identification. For validation, the HIV and FIV virus preparations were further quantified by a biochemical technique and the results obtained by both approaches corroborated well. We further demonstrate that the electrical technique could be applied to accurately measure and characterize silica nanoparticles that resemble the virus particles in size. Based on these results, we anticipate our present approach to be a starting point towards establishing the foundation for label-free electrical-based identification and quantification of an unlimited number of viruses and other nano-sized particles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Here we identify and quantitate two similar viruses, human and feline immunodeficiency viruses (HIV and FIV), suspended in a liquid medium without labeling, using a semiconductor technique. The virus count was estimated by calculating the impurities inside a defined volume by observing the change in electrical parameters. Empirically, the virus count was similar to the absolute value of the ratio of the change of the virus suspension dopant concentration relative to the mock dopant over the change in virus suspension Debye volume relative to mock Debye volume. The virus type was identified by constructing a concentration-mobility relationship which is unique for each kind of virus, allowing for a fast (within minutes) and label-free virus quantification and identification. For validation, the HIV and FIV virus preparations were further quantified by a biochemical technique and the results obtained by both approaches corroborated well. We further demonstrate that the electrical technique could be applied to accurately measure and characterize silica nanoparticles that resemble the virus particles in size. Based on these results, we anticipate our present approach to be a starting point towards establishing the foundation for label-free electrical-based identification and quantification of an unlimited number of viruses and other nano-sized particles.
2013
Aktar, S. J.; Jabeen, A.; Ali, L. M.; Vivet-Boudou, V.; Marquet, R.; Rizvi, T. A.
SHAPE analysis of the 5' end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization Journal Article
In: Rna, vol. 19, no. 12, pp. 1648-58, 2013, ISSN: 1355-8382 (Print) 1355-8382.
@article{RN80,
title = {SHAPE analysis of the 5' end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization},
author = {S. J. Aktar and A. Jabeen and L. M. Ali and V. Vivet-Boudou and R. Marquet and T. A. Rizvi},
doi = {10.1261/rna.040931.113},
issn = {1355-8382 (Print) 1355-8382},
year = {2013},
date = {2013-01-01},
urldate = {2013-01-01},
journal = {Rna},
volume = {19},
number = {12},
pages = {1648-58},
abstract = {Earlier genetic and structural prediction analyses revealed that the packaging determinants of Mason Pfizer monkey virus (MPMV) include two discontinuous core regions at the 5' end of its genomic RNA. RNA secondary structure predictions suggested that these packaging determinants fold into several stem-loops (SLs). To experimentally validate this structural model, we employed selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), which examines the flexibility of the RNA backbone at each nucleotide position. Our SHAPE data validated several predicted structural motifs, including U5/Gag long-range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive G-C-rich palindromic (pal) SL. Minimum free-energy structure predictions, phylogenetic, and in silico modeling analyses of different MPMV strains revealed that the U5 and gag sequences involved in the LRIs differ minimally within strains and maintain a very high degree of complementarity. Since the pal SL forms a helix loop containing a canonical "GC" dyad, it may act as a RNA dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Analyses of wild-type and pal mutant RNAs revealed that disruption of pal sequence strongly affected RNA dimerization. However, when in vitro transcribed trans-complementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5'-CGGCCG-3') functions as DIS.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Earlier genetic and structural prediction analyses revealed that the packaging determinants of Mason Pfizer monkey virus (MPMV) include two discontinuous core regions at the 5' end of its genomic RNA. RNA secondary structure predictions suggested that these packaging determinants fold into several stem-loops (SLs). To experimentally validate this structural model, we employed selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), which examines the flexibility of the RNA backbone at each nucleotide position. Our SHAPE data validated several predicted structural motifs, including U5/Gag long-range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive G-C-rich palindromic (pal) SL. Minimum free-energy structure predictions, phylogenetic, and in silico modeling analyses of different MPMV strains revealed that the U5 and gag sequences involved in the LRIs differ minimally within strains and maintain a very high degree of complementarity. Since the pal SL forms a helix loop containing a canonical "GC" dyad, it may act as a RNA dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Analyses of wild-type and pal mutant RNAs revealed that disruption of pal sequence strongly affected RNA dimerization. However, when in vitro transcribed trans-complementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5'-CGGCCG-3') functions as DIS.
Saeed, I. A.; Ali, L.; Jabeen, A.; Khasawneh, M.; Rizvi, T. A.; Ashraf, S. S.
Estrogenic activities of ten medicinal herbs from the Middle East Journal Article
In: J Chromatogr Sci, vol. 51, no. 1, pp. 33-9, 2013, ISSN: 0021-9665.
@article{RN54,
title = {Estrogenic activities of ten medicinal herbs from the Middle East},
author = {I. A. Saeed and L. Ali and A. Jabeen and M. Khasawneh and T. A. Rizvi and S. S. Ashraf},
doi = {10.1093/chromsci/bms101},
issn = {0021-9665},
year = {2013},
date = {2013-01-01},
urldate = {2013-01-01},
journal = {J Chromatogr Sci},
volume = {51},
number = {1},
pages = {33-9},
abstract = {Traditional medicinal plants have long been recognized as remedies and important sources of treatment for developing countries. In the present study, we report on a detailed study to quantify the presence of five known phytoestrogens in 10 widely used herbs used in the Middle East. Surprisingly some of these plants were almost devoid of tested phytoestrogens, whereas others were very rich in known phytoestrogens. For example, Hibiscus sabdariffa was found to be the richest in quercetin and daidzein, whereas Cyperus conglomeratus had the highest concentrations of kaempferol and genistein. On the other hand, Salvadora persica was almost devoid of the screened phytoestrogens. Ethanolic extracts were further tested for their proliferative activities in cell-culture using estrogen-responsive breast cancer cell lines (MCF-7) and were found to fall into three distinct groups based on their estrogenic activities. The most potent herbal extract (O. vulgare) was further fractionated and the fractions were analyzed again for phytoestrogenic content (using high-performance liquid chromatography) and proliferative activity. Our results indicate that the proliferative activities of some of the extracts and fractions are not completely attributable to the phytoestrogens screened, thus it is likely that some of these plants may have other (perhaps yet unknown) phytoestrogens.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Traditional medicinal plants have long been recognized as remedies and important sources of treatment for developing countries. In the present study, we report on a detailed study to quantify the presence of five known phytoestrogens in 10 widely used herbs used in the Middle East. Surprisingly some of these plants were almost devoid of tested phytoestrogens, whereas others were very rich in known phytoestrogens. For example, Hibiscus sabdariffa was found to be the richest in quercetin and daidzein, whereas Cyperus conglomeratus had the highest concentrations of kaempferol and genistein. On the other hand, Salvadora persica was almost devoid of the screened phytoestrogens. Ethanolic extracts were further tested for their proliferative activities in cell-culture using estrogen-responsive breast cancer cell lines (MCF-7) and were found to fall into three distinct groups based on their estrogenic activities. The most potent herbal extract (O. vulgare) was further fractionated and the fractions were analyzed again for phytoestrogenic content (using high-performance liquid chromatography) and proliferative activity. Our results indicate that the proliferative activities of some of the extracts and fractions are not completely attributable to the phytoestrogens screened, thus it is likely that some of these plants may have other (perhaps yet unknown) phytoestrogens.