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theses

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) lose the interaction with the host bromodomain and extraterminal (BET) proteins and decrease their integration preference at promoter/enhancers and transcriptional start sites and CpG islands. MLV lacking the IN TP by altering the open reading frame were infected into a tumorigenesis mice model (MYC/Runx2) to observe integration patterns and phenotypic effects. Viral passage resulted in the restoration the TP onto IN through small deletions. Mice infected with different modified MLV lacking the IN TP- coding sequence (TP-), showed an improved median survival by 10 days compared to wildtype (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv-20, were identified in seven mice, displaying both fast and slow tumorigenesis. Next generation sequencing of tumors showed an infected mouse (TP-16) without observed recombination with ERVs with less integrations at TSS and CpG islands, compared to the mean integrations of WT tumors. This mouse also has less integrations at Brd4 and BET associated histone modifications (H3K4me1/3) +/- 1 kb from ChIP-seq peaks. However, this mouse succumbed to the tumor rapidly (34 days). Analysis of the four of the top copy number integrants of the TP-16 tumor revealed their proximity to known MLV common insertion sites genes (Hdac6, Ccnd1, Rasgrp1), maintaining their MLV IN TP- genotype. Furthermore, mapping integrations in K562 cells revealed the preference of MLV IN TP- insertions within chromatin profile states associated with heterochromatin and weakly transcribed regions. A decreased number of integrations were observed at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). The results highlight the strong selection within the mouse to maintain the full-length IN protein. MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model. However, MLV integrations, in the absence of the influence of BET proteins, can still occur at regions of oncogenesis driver genes, either stochastically or through trans-complementation by functional endogenous Gag-Pol. Thus, the modified MLV virus can be a safer vector than the wildtype virus, but it still maintains the oncogenic potential. This study provides new insights on how to improve the safety of MLV retroviral vectors.

ETD_9818

Ph.D.

Includes bibliographical references

doi:10.7282/t3-x866-nk26

ETD doctoral

The author owns the copyright to this work.