Date of Defense
12-11-2024 1:00 PM
Location
Yannah Theater, Second Floor, Block C (2C010), Male Side
Document Type
Dissertation Defense
Degree Name
Doctor of Philosophy (PhD)
College
CMHS
Department
Medical Microbiology and Immunology
First Advisor
Prof. Tahir A. Rizvi
Abstract
Mouse mammary tumor virus (MMTV) is a betaretrovirus that induces breast cancer and sporadically, T-cell lymphomas in mice. MMTV serves as a pertinent model for elucidating the mechanism(s) of oncogenesis and the genetics associated with the development of mammary tumors. Being a rodent retrovirus, MMTV is phylogenetically distant from human and other primate retroviruses; thus, positioning it ideally as a potential vector of choice for human gene therapy. This may circumvent safety concerns such as recombination with other related endogenous human retroviruses. In addition, MMTV is distinct from most other retroviruses, but similar to lentiviruses, in that it has the ability to infect non-dividing cells. Furthermore, MMTV contains multiple steroid responsive promoters, making it not only “switchable” but also “tissue-specific” since they turn on gene expression only in cells with receptors for steroid hormones. These unique features have led to increasing interest in developing MMTV-based vectors for “targeted” and “inducible” human gene therapy, especially for breast tissue. Therefore, it is crucial that the molecular mechanisms of the pertinent aspects of MMTV life cycle be better understood to develop safer, more efficient vectors for gene delivery.
Earlier studies have suggested that MMTV harbors sequences known as packaging sequences (Psi) responsible for genomic RNA (gRNA) packaging at the 5’ end of its genome. This region has been predicted to assume a higher order structure which was validated biochemically employing hSHAPE (high throughput selective 2' hydroxyl acylation analyzed by primer extension) and shown to have four RNA-RNA long range interactions (LRIs) harboring complementary sequences. Three of these LRIs involve complementary sequences between the U5/Gag (LRIs I-III), while one LRI (LRI-IV) involves complementary sequences within U5/U5 region that are 291 and 190 nucleotides apart, respectively.
In addition to Psi, the packaging of gRNA into retroviral particles relies on its specific recognition by the Gag precursor. 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. In this study, using MMTV as a model, we investigated the effects of mutations in the four proposed LRIs on the gRNA structure and function. Our findings reveal the presence of an unpredicted extended LRI, and hSHAPE showed that maintaining a Psi structure like the wild type is crucial for efficient RNA 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. Further footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to two specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, these results reveal that the three-dimensional structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNA species that may also bind Gag efficiently
Included in
MOUSE MAMMARY TUMOR VIRUS (MMTV) GENOMIC RNA (GRNA) PACKAGING: SELECTIVE GRNA ENCAPSIDATION IS FACILITATED BY LONG-RANGE INTERACTIONS (LRIS) THAT FACILITATE SPECIFIC GAG BINDING
Yannah Theater, Second Floor, Block C (2C010), Male Side
Mouse mammary tumor virus (MMTV) is a betaretrovirus that induces breast cancer and sporadically, T-cell lymphomas in mice. MMTV serves as a pertinent model for elucidating the mechanism(s) of oncogenesis and the genetics associated with the development of mammary tumors. Being a rodent retrovirus, MMTV is phylogenetically distant from human and other primate retroviruses; thus, positioning it ideally as a potential vector of choice for human gene therapy. This may circumvent safety concerns such as recombination with other related endogenous human retroviruses. In addition, MMTV is distinct from most other retroviruses, but similar to lentiviruses, in that it has the ability to infect non-dividing cells. Furthermore, MMTV contains multiple steroid responsive promoters, making it not only “switchable” but also “tissue-specific” since they turn on gene expression only in cells with receptors for steroid hormones. These unique features have led to increasing interest in developing MMTV-based vectors for “targeted” and “inducible” human gene therapy, especially for breast tissue. Therefore, it is crucial that the molecular mechanisms of the pertinent aspects of MMTV life cycle be better understood to develop safer, more efficient vectors for gene delivery.
Earlier studies have suggested that MMTV harbors sequences known as packaging sequences (Psi) responsible for genomic RNA (gRNA) packaging at the 5’ end of its genome. This region has been predicted to assume a higher order structure which was validated biochemically employing hSHAPE (high throughput selective 2' hydroxyl acylation analyzed by primer extension) and shown to have four RNA-RNA long range interactions (LRIs) harboring complementary sequences. Three of these LRIs involve complementary sequences between the U5/Gag (LRIs I-III), while one LRI (LRI-IV) involves complementary sequences within U5/U5 region that are 291 and 190 nucleotides apart, respectively.
In addition to Psi, the packaging of gRNA into retroviral particles relies on its specific recognition by the Gag precursor. 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. In this study, using MMTV as a model, we investigated the effects of mutations in the four proposed LRIs on the gRNA structure and function. Our findings reveal the presence of an unpredicted extended LRI, and hSHAPE showed that maintaining a Psi structure like the wild type is crucial for efficient RNA 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. Further footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to two specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, these results reveal that the three-dimensional structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNA species that may also bind Gag efficiently