COVID-19 is currently a highly pressing health threat and therapeutic strategies to mitigate the infection impact are urgently needed . Characterization of the SARS-CoV-2 interactome in infected cells may represent a powerful tool to identify cellular proteins hijacked by viruses for their life cycle and develop host-oriented antiviral therapeutics . Here we report the proteomic characterization of host proteins interacting with SARS-CoV-2 Nucleoprotein in infected Vero E6 cells . We identified 24 high-confidence proteins mainly playing a role in RNA metabolism and translation, including RNA helicases and scaffold proteins involved in the formation of stress granules, cytoplasmic aggregates of messenger ribonucleoproteins that accumulate as a result of stress-induced translation arrest . Analysis of stress granules upon SARS-CoV-2 infection showed that these structures are not induced in infected cells, neither eIF2α phosphorylation, an upstream event leading to stress-induced translation inhibition . Notably, we found that G3BP1, a stress granule component that associates with the Nucleoprotein, is required for efficient SARS-CoV-2 replication . Moreover, we showed that the Nucleoprotein-interacting RNA helicase DDX3X colocalizes with viral RNA foci and its inhibition by small molecules or small interfering RNAs significantly reduces viral replication . Altogether, these results indicate that SARS-CoV-2 subverts the stress granule machinery and exploits G3BP1 and DDX3X for its replication cycle, offering groundwork for future development of host-directed therapies.