RESUMO
Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA potentially through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which may lead to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.
Assuntos
Vírus da Influenza A , Influenza Humana , RNA Mensageiro , RNA Viral , Proteínas de Ligação a RNA , Animais , Humanos , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Vírus da Influenza A/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Influenza Humana/genética , Transporte de RNA , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Viral/metabolismo , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Replicação ViralRESUMO
Genome-wide approaches have significantly advanced our knowledge of the repertoire of RNA-binding proteins (RBPs) that associate with cellular polyadenylated mRNAs within eukaryotic cells. Recent studies focusing on the RBP interactomes of viral mRNAs, notably SARS-Cov-2, have revealed both similarities and differences between the RBP profiles of viral and cellular mRNAs. However, the RBPome of influenza virus mRNAs remains unexplored. Herein, we identify RBPs that associate with the viral mRNA encoding the nucleoprotein (NP) of an influenza A virus. Focusing on TDP-43, we show that it binds several influenza mRNAs beyond the NP-mRNA, and that its depletion results in lower levels of viral mRNAs and proteins within infected cells, and a decreased yield of infectious viral particles. We provide evidence that the viral polymerase recruits TDP-43 onto viral mRNAs through a direct interaction with the disordered C-terminal domain of TDP-43. Notably, other RBPs found to be associated with influenza virus mRNAs also interact with the viral polymerase, which points to a role of the polymerase in orchestrating the assembly of viral messenger ribonucleoproteins.
Assuntos
Proteínas de Ligação a DNA , Vírus da Influenza A , RNA Mensageiro , RNA Viral , Proteínas de Ligação a RNA , Replicação Viral , Humanos , Replicação Viral/genética , RNA Viral/metabolismo , RNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Vírus da Influenza A/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Células HEK293 , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/genética , Ligação Proteica , AnimaisRESUMO
Human cytomegalovirus (HCMV) replicates its DNA genome in specialized replication compartments (RCs) in the host cell nucleus. These membrane-less organelles originate as spherical structures and grow in size over time. However, the mechanism of RC biogenesis has remained understudied. Using live-cell imaging and photo-oligomerization, we show that a central component of RCs, the UL112-113 proteins, undergo liquid-liquid phase separation (LLPS) to form RCs in the nucleus. We show that the self-interacting domain and large intrinsically disordered regions of UL112-113 are required for LLPS. Importantly, viral DNA induces local clustering of these proteins and lowers the threshold for phase separation. The formation of phase-separated compartments around viral genomes is necessary to recruit the viral DNA polymerase for viral genome replication. Thus, HCMV uses its UL112-113 proteins to generate RCs around viral genomes by LLPS to ensure the formation of a pro-replicative environment.