Significance Alphaviruses are mosquito-borne RNA viruses that cause rash, arthritis, and neurologic disease. Despite a continued risk of outbreaks, there are no licensed interventions for any alphavirus. For progress in control, an understanding of the molecular targets that affect virus replication and virulence is essential. This paper characterizes a conserved macrodomain in the virulence factor nonstructural protein 3 (nsP3). We discovered that the macrodomain ADP-ribosylhydrolase activity is critical for controlling the composition of cellular condensates, specifically through regulating the localization of translation factors, during viral infection. Given that this macrodomain is conserved across alphaviruses and coronaviruses and that the associated enzymatic activity is critical for virulence, our work may open avenues for developing a class of antiviral therapeutics.