RESUMO
Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.
Assuntos
HIV-1/genética , HIV-1/metabolismo , Edição de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Regiões 3' não Traduzidas , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Clonagem Molecular , Regulação Viral da Expressão Gênica , Genoma Viral , Células HEK293 , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
High-risk human papillomaviruses (HPVs), including HPV-16 and HPV-18, are the causative agents of cervical carcinomas and are linked to several other tumors of the anogenital and oropharyngeal regions. The majority of HPV-induced tumors contain integrated copies of the normally episomal HPV genome that invariably retain intact forms of the two HPV oncogenes E6 and E7. E6 induces degradation of the cellular tumor suppressor p53, while E7 destabilizes the retinoblastoma (Rb) protein. Previous work has shown that loss of E6 function in cervical cancer cells induces p53 expression as well as downstream effectors that induce apoptosis and cell cycle arrest. Similarly, loss of E7 allows increased Rb expression, leading to cell cycle arrest and senescence. Here, we demonstrate that expression of a bacterial Cas9 RNA-guided endonuclease, together with single guide RNAs (sgRNAs) specific for E6 or E7, is able to induce cleavage of the HPV genome, resulting in the introduction of inactivating deletion and insertion mutations into the E6 or E7 gene. This results in the induction of p53 or Rb, leading to cell cycle arrest and eventual cell death. Both HPV-16- and HPV-18-transformed cells were found to be responsive to targeted HPV genome-specific DNA cleavage. These data provide a proof of principle for the idea that vector-delivered Cas9/sgRNA combinations could represent effective treatment modalities for HPV-induced cancers. Importance: Human papillomaviruses (HPVs) are the causative agents of almost all cervical carcinomas and many other tumors, including many head and neck cancers. In these cancer cells, the HPV DNA genome is integrated into the cellular genome, where it expresses high levels of two viral oncogenes, called E6 and E7, that are required for cancer cell growth and viability. Here, we demonstrate that the recently described bacterial CRISPR/Cas RNA-guided endonuclease can be reprogrammed to target and destroy the E6 or E7 gene in cervical carcinoma cells transformed by HPV, resulting in cell cycle arrest, leading to cancer cell death. We propose that viral vectors designed to deliver E6- and/or E7-specific CRISPR/Cas to tumor cells could represent a novel and highly effective tool to treat and eliminate HPV-induced cancers.