RNA polymerase III (Pol III) transcribes  transfer RNAs (tRNAs) and other non-coding RNAs (ncRNAs) essential to cellular function. Additionally, Pol III serves to support DNA damage responses while cytosolic forms act as DNA immune sensors that produce substrates for sensing by RIG-I to activate IFN pathways. Genetic mutations in the subunits of Pol III are associated with a spectrum of neurodegenerative diseases and increased susceptibility to severe herpesvirus infections. However, many aspects of Pol III transcription and processing remain poorly understood, mainly due to a lack of available sensitive and systematic methods for their analysis.  To address this, we developed DRAP3R (Direct Read and Analysis of Polymerase III transcribed RNAs), a modified nanopore direct RNA sequencing approach and analysis framework that enables the specific and sensitive capture of nascent Pol III transcribed RNAs. Applying DRAP3R to distinct cell types, we identified novel Pol III RNAs, thus expanding the known Pol III transcriptome, while also developing downstream analytical approaches to robustly identify RNA modifications such as pseudouridine (Ψ) and N6-methyladenosine (m6A) at nucleotide-level resolution on individual RNAs.  Application of DRAP3R to measure how diverse viruses regulate the Pol III transcriptome/epitranscriptome in infected cells enables our long-term aim of identifying the molecular pathways targeted during infection. Further, by combining DRAP3R with nano-tRNA-Seq, we determine the impacts of viral infection at different stages of tRNA maturation. In this presentation, I will demonstrate how these approaches provide a new window into an underappreciated and understudied area of viral infection.