Our Technophile
Fathima N P
Past PhD Student
Fathima Nuzra Nagoor Pitchai (2016-2021): Defended her PhD thesis entitled, “Delineating the binding sites of Mason-Pfizer monkey virus (MPMV) Gag precursor polyprotein (Pr78Gag) on genomic RNA for its selective packaging”. Her thesis work was directed towards expressing, purifying, and characterizing MPMV Pr78Gag, characterizing Pr78Gag binding site(s) on the MPMV gRNA, and establishing correlations between the Pr78Gag binding site(s) and gRNA packaging (Pitchai et al., Sci. Rep., 2018; Pitchai et al., J. Mol. Biol., 2021). In addition, she contributed significantly to other projects in the laboratory to be a co-author on other manuscripts (Chameettachal et al., Viruses, 2018; Krishnan et al., Viruses, 2019; Ali et al., Front. Micro., 2020; Chameettachal et al., Nucleic Acids Research, 2021; Pillai et al., J. Mol. Biol., 2021). Finally, she also gave a talk at the prestigious annual Retrovirus meeting of the Cold Spring Harbor Laboratory, New York, 2020 (https://youtu.be/L41Icadeiek). Fatima is currently working as a postdoctoral fellow at the Gladstone Institute of Virology, University of California-San Francisco (UCSF), USA. Link to the thesis defense.
Publications
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.
2018
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.
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.