Caracterização molecular da infecção por Zika Vírus (ZIKV) em Glioblastoma (GBM)

Abstract

Glioblastoma (GBM) is a malignant tumor arising from astrocytic differentiation. With a mean survival of only two years after diagnosis, it is the most common and aggressive primary brain tumor, accounting for 80% of malignant neoplasms of the central nervous system (CNS). In the absence of curative treatment, current therapeutic strategies rely on surgical resection followed by chemo- and radiotherapy combined with palliative care. Given this limited scenario, targeted therapies have emerged as promising alternatives, including the search for immunotherapeutic targets, such as the membrane receptor PTPRZ1, implicated in GBM development, and the investigation of oncolytic viruses capable of selectively infecting and destroying tumor cells. Advancing such approaches requires a detailed understanding of virus–tumor interactions. The aim of this thesis was to characterize the genes, cellular pathways, and epitranscriptomic factors modulated by Zika virus (ZIKV; Flavivirus, Flaviviridae) infection in GBM cells. In Chapter 1, a systematic review and functional meta-analysis of biological pathways was conducted for studies published up to March 5th, 2025, characterizing the transcriptomic profile of ZIKV-infected GBM cells. Of the 139 records identified, five met the eligibility criteria (open-access primary studies assessing differentially expressed genes - DEGs - in GBM models infected with wild-type or engineered ZIKV using transcriptomic approaches). Across studies, 4,360 genes were reported as upregulated and 2,072 as downregulated, with twelve genes (DNAJB9, SESN2, PMAIP1, PPP1R15A, KLF4, ATF3, IFNB1, IFNL1, ANKRD33B, ZC3HAV1, OASL, and CCL5) consistently overexpressed in all datasets. Functional analysis revealed 23 commonly activated pathways, predominantly linked to innate immune responses mediated by type I and type III interferons. However, methodological heterogeneity and incomplete reporting limited the comparability across studies, underscoring the need for standardization. In Chapter 2, the transcriptome and epitranscriptome of the U-87MG cell line infected with the ZIKV strain PE243 were characterized using direct RNA sequencing. Twenty-one DEGs were identified, being 17: IFIT2, CCL5, RSAD2, CXCL10, OASL, CXCL8, H3C4, CXCL11, IFIT1, RAET1L, IL7R, CXCL1, IFNL1, H4C16, HLA-DOB, ACTN2, RND1, EXOSC4, and the RNA ENSG00000286610 all upregulated. Only the gene CCDC85A and the RNA RN7SL731P appeared as downregulated. A total of 1,302 differentially expressed transcripts were detected, a number 64-fold greater than the DEGs, highlighting the central role of alternative splicing in shaping the cellular response to infection. Epitranscriptomic analyses revealed, for the first time in GBM cells, ZIKV-induced changes in N6-methyladenosine (m6A), 5-methylcytosine (m⁵C), pseudouridine (pseU), and inosine (I). Several genes exhibited concordant isoform-level regulation: RPLP1, RPL15, and RPL8 were consistently upregulated, indicating coordinated modulation of ribosomal components and suggesting ZIKV-induced translational reprogramming. In Chapter 3, the role of the cellular receptor PTPRZ1, expressed in GBM and associated with tumor prognosis, was investigated in the context of ZIKV infection. A viral kinetics assay with RT-qPCR quantification initially suggested transcriptional induction of PTPRZ1 during infection; however, subsequent blocking and competition experiments excluded this receptor as a mediator of viral entry or infectivity in GBM models. Taken together, the findings of this thesis advance the understanding of the molecular mechanisms underlying the interaction between ZIKV and GBM, revealing robust alterations in the transcriptome and epitranscriptome, coupled with activation of immune pathways, without evidence supporting a functional role for PTPRZ1 in this context. These results provide a foundation for future research aimed at identifying candidate targets for ZIKV-based oncolytic strategies in glioblastoma therapy.