Our Technophile
Pretty P
Past Research Staff
Pretty P (2002-2010): Pretty Phillip was the first hire when Rizvi Lab moved to the UAE in 2002. She worked in this laboratory till 2010 with exceptional dedication. She was instrumental in establishing the laboratory from the ground up, and without her support, it would not have developed into what it is today. In 2010, she chose to pursue a more permanent position at the College of Medicine as a Medical Research Specialist.
Publications
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.
2011
Shamsi, I. R. Al; Dhaheri, N. S. Al; Phillip, P. S.; Mustafa, F.; Rizvi, T. A.
Reciprocal cross-packaging of primate lentiviral (HIV-1 and SIV) RNAs by heterologous non-lentiviral MPMV proteins Journal Article
In: Virus Res, vol. 155, no. 1, pp. 352-7, 2011, ISSN: 0168-1702.
@article{RN70,
title = {Reciprocal cross-packaging of primate lentiviral (HIV-1 and SIV) RNAs by heterologous non-lentiviral MPMV proteins},
author = {I. R. Al Shamsi and N. S. Al Dhaheri and P. S. Phillip and F. Mustafa and T. A. Rizvi},
doi = {10.1016/j.virusres.2010.09.018},
issn = {0168-1702},
year = {2011},
date = {2011-01-01},
urldate = {2011-01-01},
journal = {Virus Res},
volume = {155},
number = {1},
pages = {352-7},
abstract = {Retroviral RNA packaging signal (ψ) allows the preferential packaging of genomic RNA into virus particles through its interaction with the nucleocapsid protein. The specificity of this interaction came into question when it was shown that primate retroviruses, such as HIV-1, could cross-package RNA from its simian cousin, SIV, and vice versa and that feline retrovirus, FIV could cross-package RNA from a distantly related primate retrovirus, MPMV. To study the generality of this phenomenon further, we determined whether there is a greater packaging restriction between the lentiviral class of retroviruses (HIV-1 and SIV) and a non-lentivirus, MPMV. Our results revealed that primate lentiviral RNAs can be cross-packaged by primate non-lentiviral particles reciprocally, but the cross-packaged RNAs could not be propagated by the heterologous particles. Packaging of RNA in the context of both retroviral vectors as well as non-retroviral RNA containing SIV, HIV, and MPMV packaging determinants by each others proteins further confirmed the specificity of cross-packaging conferred by the packaging sequences. These results reveal the promiscuous nature of retroviral packaging determinants and raise caution against their wide spread presence on retroviral vectors to be used for human gene therapy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Retroviral RNA packaging signal (ψ) allows the preferential packaging of genomic RNA into virus particles through its interaction with the nucleocapsid protein. The specificity of this interaction came into question when it was shown that primate retroviruses, such as HIV-1, could cross-package RNA from its simian cousin, SIV, and vice versa and that feline retrovirus, FIV could cross-package RNA from a distantly related primate retrovirus, MPMV. To study the generality of this phenomenon further, we determined whether there is a greater packaging restriction between the lentiviral class of retroviruses (HIV-1 and SIV) and a non-lentivirus, MPMV. Our results revealed that primate lentiviral RNAs can be cross-packaged by primate non-lentiviral particles reciprocally, but the cross-packaged RNAs could not be propagated by the heterologous particles. Packaging of RNA in the context of both retroviral vectors as well as non-retroviral RNA containing SIV, HIV, and MPMV packaging determinants by each others proteins further confirmed the specificity of cross-packaging conferred by the packaging sequences. These results reveal the promiscuous nature of retroviral packaging determinants and raise caution against their wide spread presence on retroviral vectors to be used for human gene therapy.
Kenyon, J. C.; Tanner, S. J.; Legiewicz, M.; Phillip, P. S.; Rizvi, T. A.; Grice, S. F. Le; Lever, A. M.
SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization Journal Article
In: Nucleic Acids Res, vol. 39, no. 15, pp. 6692-704, 2011, ISSN: 0305-1048 (Print) 0305-1048.
@article{RN67,
title = {SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization},
author = {J. C. Kenyon and S. J. Tanner and M. Legiewicz and P. S. Phillip and T. A. Rizvi and S. F. Le Grice and A. M. Lever},
doi = {10.1093/nar/gkr252},
issn = {0305-1048 (Print) 0305-1048},
year = {2011},
date = {2011-01-01},
urldate = {2011-01-01},
journal = {Nucleic Acids Res},
volume = {39},
number = {15},
pages = {6692-704},
abstract = {Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.
2010
Jaballah, S. A.; Aktar, S. J.; Ali, J.; Phillip, P. S.; Dhaheri, N. S. Al; Jabeen, A.; Rizvi, T. A.
In: J Mol Biol, vol. 401, no. 5, pp. 996-1014, 2010, ISSN: 0022-2836.
@article{RN42,
title = {A G-C-rich palindromic structural motif and a stretch of single-stranded purines are required for optimal packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA},
author = {S. A. Jaballah and S. J. Aktar and J. Ali and P. S. Phillip and N. S. Al Dhaheri and A. Jabeen and T. A. Rizvi},
doi = {10.1016/j.jmb.2010.06.043},
issn = {0022-2836},
year = {2010},
date = {2010-01-01},
urldate = {2010-01-01},
journal = {J Mol Biol},
volume = {401},
number = {5},
pages = {996-1014},
abstract = {During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5' end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5' untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region "A", immediately downstream of the primer binding site, is composed of 50 nt, whereas region "B" is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5' end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5' untranslated region (UTR) and Gag. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region "A", immediately downstream of the primer binding site, is composed of 50 nt, whereas region "B" is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process.
Rizvi, T. A.; Kenyon, J. C.; Ali, J.; Aktar, S. J.; Phillip, P. S.; Ghazawi, A.; Mustafa, F.; Lever, A. M. L.
Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag Journal Article
In: J Mol Biol, vol. 403, no. 1, pp. 103-119, 2010, ISSN: 0022-2836 (Print) 0022-2836.
@article{RN49,
title = {Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag},
author = {T. A. Rizvi and J. C. Kenyon and J. Ali and S. J. Aktar and P. S. Phillip and A. Ghazawi and F. Mustafa and A. M. L. Lever},
doi = {10.1016/j.jmb.2010.08.019},
issn = {0022-2836 (Print) 0022-2836},
year = {2010},
date = {2010-01-01},
urldate = {2010-01-01},
journal = {J Mol Biol},
volume = {403},
number = {1},
pages = {103-119},
abstract = {The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.
2009
Rizvi, T. A.; Ali, J.; Phillip, P. S.; Ghazawi, A.; Jayanth, P.; Mustafa, F.
In: Virology, vol. 385, no. 2, pp. 464-72, 2009, ISSN: 0042-6822.
@article{RN94,
title = {Role of a heterologous retroviral transport element in the development of genetic complementation assay for mouse mammary tumor virus (MMTV) replication},
author = {T. A. Rizvi and J. Ali and P. S. Phillip and A. Ghazawi and P. Jayanth and F. Mustafa},
doi = {10.1016/j.virol.2008.12.027},
issn = {0042-6822},
year = {2009},
date = {2009-01-01},
urldate = {2009-01-01},
journal = {Virology},
volume = {385},
number = {2},
pages = {464-72},
abstract = {The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.
Dhaheri, N. S. Al; Phillip, P. S.; Ghazawi, A.; Ali, J.; Beebi, E.; Jaballah, S. A.; Rizvi, T. A.
Cross-packaging of genetically distinct mouse and primate retroviral RNAs Journal Article
In: Retrovirology, vol. 6, pp. 66, 2009, ISSN: 1742-4690.
@article{RN66,
title = {Cross-packaging of genetically distinct mouse and primate retroviral RNAs},
author = {N. S. Al Dhaheri and P. S. Phillip and A. Ghazawi and J. Ali and E. Beebi and S. A. Jaballah and T. A. Rizvi},
doi = {10.1186/1742-4690-6-66},
issn = {1742-4690},
year = {2009},
date = {2009-01-01},
urldate = {2009-01-01},
journal = {Retrovirology},
volume = {6},
pages = {66},
abstract = {BACKGROUND: The mouse mammary tumor virus (MMTV) is unique from other retroviruses in having multiple viral promoters, which can be regulated by hormones in a tissue specific manner. This unique property has lead to increased interest in studying MMTV replication with the hope of developing MMTV based vectors for human gene therapy. However, it has recently been reported that related as well as unrelated retroviruses can cross-package each other's genome raising safety concerns towards the use of candidate retroviral vectors for human gene therapy. Therefore, using a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV) that has intracellular assembly process similar to that of MMTV. RESULTS: Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the heterologous virus particles reciprocally suggesting that pseudotyping between two genetically distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To further confirm that the specificity of cross-packaging was conferred by the packaging sequences (psi), we cloned the packaging sequences of these viruses on expression plasmids that generated non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was primarily due to the recognition of psi by the heterologous virus proteins. CONCLUSION: The results presented in this study strongly argue that MPMV and MMTV are promiscuous in their ability to cross-package each other's genome suggesting potential RNA-protein interactions among divergent retroviral RNAs proposing that these interactions are more complicated than originally thought. Furthermore, these observations raise the possibility that MMTV and MPMV genomes could also co-package providing substrates for exchanging genetic information.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: The mouse mammary tumor virus (MMTV) is unique from other retroviruses in having multiple viral promoters, which can be regulated by hormones in a tissue specific manner. This unique property has lead to increased interest in studying MMTV replication with the hope of developing MMTV based vectors for human gene therapy. However, it has recently been reported that related as well as unrelated retroviruses can cross-package each other's genome raising safety concerns towards the use of candidate retroviral vectors for human gene therapy. Therefore, using a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV) that has intracellular assembly process similar to that of MMTV. RESULTS: Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the heterologous virus particles reciprocally suggesting that pseudotyping between two genetically distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To further confirm that the specificity of cross-packaging was conferred by the packaging sequences (psi), we cloned the packaging sequences of these viruses on expression plasmids that generated non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was primarily due to the recognition of psi by the heterologous virus proteins. CONCLUSION: The results presented in this study strongly argue that MPMV and MMTV are promiscuous in their ability to cross-package each other's genome suggesting potential RNA-protein interactions among divergent retroviral RNAs proposing that these interactions are more complicated than originally thought. Furthermore, these observations raise the possibility that MMTV and MPMV genomes could also co-package providing substrates for exchanging genetic information.
2008
Kenyon, J. C.; Ghazawi, A.; Cheung, W. K.; Phillip, P. S.; Rizvi, T. A.; Lever, A. M.
In: Rna, vol. 14, no. 12, pp. 2597-608, 2008, ISSN: 1355-8382 (Print) 1355-8382.
@article{RN60,
title = {The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop},
author = {J. C. Kenyon and A. Ghazawi and W. K. Cheung and P. S. Phillip and T. A. Rizvi and A. M. Lever},
doi = {10.1261/rna.1284908},
issn = {1355-8382 (Print) 1355-8382},
year = {2008},
date = {2008-01-01},
urldate = {2008-01-01},
journal = {Rna},
volume = {14},
number = {12},
pages = {2597-608},
abstract = {Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.
2006
Ghazawi, A.; Mustafa, F.; Phillip, P. S.; Jayanth, P.; Ali, J.; Rizvi, T. A.
In: Microbes Infect, vol. 8, no. 3, pp. 767-78, 2006, ISSN: 1286-4579 (Print) 1286-4579.
@article{RN118,
title = {Both the 5' and 3' LTRs of FIV contain minor RNA encapsidation determinants compared to the two core packaging determinants within the 5' untranslated region and gag},
author = {A. Ghazawi and F. Mustafa and P. S. Phillip and P. Jayanth and J. Ali and T. A. Rizvi},
doi = {10.1016/j.micinf.2005.09.015},
issn = {1286-4579 (Print) 1286-4579},
year = {2006},
date = {2006-01-01},
urldate = {2006-01-01},
journal = {Microbes Infect},
volume = {8},
number = {3},
pages = {767-78},
abstract = {This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.
2005
Mustafa, F.; Ghazawi, A.; Jayanth, P.; Phillip, P. S.; Ali, J.; Rizvi, T. A.
In: J Virol, vol. 79, no. 21, pp. 13817-21, 2005, ISSN: 0022-538X (Print) 0022-538x.
@article{RN121,
title = {Sequences intervening between the core packaging determinants are dispensable for maintaining the packaging potential and propagation of feline immunodeficiency virus transfer vector RNAs},
author = {F. Mustafa and A. Ghazawi and P. Jayanth and P. S. Phillip and J. Ali and T. A. Rizvi},
doi = {10.1128/jvi.79.21.13817-13821.2005},
issn = {0022-538X (Print) 0022-538x},
year = {2005},
date = {2005-01-01},
urldate = {2005-01-01},
journal = {J Virol},
volume = {79},
number = {21},
pages = {13817-21},
abstract = {The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to approximately 150 bp of the 5' untranslated region and the first approximately 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to approximately 150 bp of the 5' untranslated region and the first approximately 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.
Mustafa, F.; Jayanth, P.; Phillip, P. S.; Ghazawi, A.; Schmidt, R. D.; Lew, K. A.; Rizvi, T. A.
Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines Journal Article
In: Microbes Infect, vol. 7, no. 2, pp. 233-9, 2005, ISSN: 1286-4579 (Print) 1286-4579.
@article{RN139,
title = {Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines},
author = {F. Mustafa and P. Jayanth and P. S. Phillip and A. Ghazawi and R. D. Schmidt and K. A. Lew and T. A. Rizvi},
doi = {10.1016/j.micinf.2004.10.015},
issn = {1286-4579 (Print) 1286-4579},
year = {2005},
date = {2005-01-01},
urldate = {2005-01-01},
journal = {Microbes Infect},
volume = {7},
number = {2},
pages = {233-9},
abstract = {The feline immunodeficiency virus (FIV) long terminal repeat (LTR), compared with some primate lentiviral LTRs, is quite a strong basal promoter. However, it seems to be highly species-specific in function and generally not very efficient in cells of non-feline origin. This study systematically explored the function of the FIV LTR in simian Cos cells compared with its activity in feline and human cells. Our studies, using biologically relevant two- and three-plasmid trans complementation assays followed by semi-quantitative reverse transcriptase PCR, show that the FIV LTR is functional in Cos cells. The results of the Cos experiment are different from previously reported literature and suggest that the strain specificity of the FIV LTR is an important determinant of whether the LTR will be functional in a particular cell type.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The feline immunodeficiency virus (FIV) long terminal repeat (LTR), compared with some primate lentiviral LTRs, is quite a strong basal promoter. However, it seems to be highly species-specific in function and generally not very efficient in cells of non-feline origin. This study systematically explored the function of the FIV LTR in simian Cos cells compared with its activity in feline and human cells. Our studies, using biologically relevant two- and three-plasmid trans complementation assays followed by semi-quantitative reverse transcriptase PCR, show that the FIV LTR is functional in Cos cells. The results of the Cos experiment are different from previously reported literature and suggest that the strain specificity of the FIV LTR is an important determinant of whether the LTR will be functional in a particular cell type.
2004
Mustafa, F.; Phillip, P. S.; Jayanth, P.; Ghazawi, A.; Lew, K. A.; Schmidt, R. D.; Rizvi, T. A.
Close proximity of the MPMV CTE to the polyadenylation sequences is important for efficient function in the subgenomic context Journal Article
In: Virus Res, vol. 105, no. 2, pp. 209-18, 2004, ISSN: 0168-1702 (Print) 0168-1702.
@article{RN143,
title = {Close proximity of the MPMV CTE to the polyadenylation sequences is important for efficient function in the subgenomic context},
author = {F. Mustafa and P. S. Phillip and P. Jayanth and A. Ghazawi and K. A. Lew and R. D. Schmidt and T. A. Rizvi},
doi = {10.1016/j.virusres.2004.06.014},
issn = {0168-1702 (Print) 0168-1702},
year = {2004},
date = {2004-01-01},
urldate = {2004-01-01},
journal = {Virus Res},
volume = {105},
number = {2},
pages = {209-18},
abstract = {The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) is a short cis-acting sequence element critical for virus gene expression. Analogous to the Rev/Rev Responsive Element (RRE) of primate lentiviruses, CTE allows the nucleocytoplasmic transport of unspliced viral mRNAs. In fact, CTE can functionally replace Rev/RRE in the genomic context and has been used successfully in the expression of viral and cellular genes from expression vectors as well. However, unlike RRE, CTE accomplishes this by interacting with cellular factors, making CTE function independent of co-expressed trans factors. Thus, CTE has proven to be a valuable tool in the expression of heterologous genes. Our previous studies have shown that close proximity of CTE to the polyadenylation sequences is important for CTE function in the genomic context. However, it is controversial whether CTE needs to be located spatially close to the polyadenylation sequences in the subgenomic context. Since CTE is being frequently used in expression vectors, we investigated the position dependency of CTE in the heterologous, subgenomic background using both genetic and structural analyses. Our results reveal that similar to the genomic situation, close proximity of CTE to the polyadenylation sequences is important for its function in the heterologous subgenomic context.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) is a short cis-acting sequence element critical for virus gene expression. Analogous to the Rev/Rev Responsive Element (RRE) of primate lentiviruses, CTE allows the nucleocytoplasmic transport of unspliced viral mRNAs. In fact, CTE can functionally replace Rev/RRE in the genomic context and has been used successfully in the expression of viral and cellular genes from expression vectors as well. However, unlike RRE, CTE accomplishes this by interacting with cellular factors, making CTE function independent of co-expressed trans factors. Thus, CTE has proven to be a valuable tool in the expression of heterologous genes. Our previous studies have shown that close proximity of CTE to the polyadenylation sequences is important for CTE function in the genomic context. However, it is controversial whether CTE needs to be located spatially close to the polyadenylation sequences in the subgenomic context. Since CTE is being frequently used in expression vectors, we investigated the position dependency of CTE in the heterologous, subgenomic background using both genetic and structural analyses. Our results reveal that similar to the genomic situation, close proximity of CTE to the polyadenylation sequences is important for its function in the heterologous subgenomic context.