Essentiality and dynamic expression of the human tRNA pool during viral infection [CRISPRlib]

Human viruses rely on host translation resources, including the cellular tRNA pool, because they lack tRNA genes. Using tRNA sequencing, we profiled mature tRNAs during infections with human cytomegalovirus (HCMV) and SARS-CoV-2. HCMV-induced alterations in mature tRNA levels were predominantly virus-driven, with minimal influence from the cellular immune response. Certain post-transcriptional modifications correlated with tRNA stability and were actively manipulated by HCMV. By contrast, SARS-CoV-2 caused minimal changes in mature tRNA levels or modifications. Comparing viral codon usage with proliferation- versus differentiation-associated codon-usage signatures in human genes revealed striking divergence. HCMV genes aligned with differentiation codon usage, whereas SARS-CoV-2 genes matched proliferation codon usage. Structural and gene-expression genes in both viruses showed strong adaptation to host tRNA pools. Finally, a systematic CRISPR screen of human tRNA genes and tRNA-modifying enzymes identified specific tRNAs and enzymes that either enhanced or restricted HCMV infectivity and influenced cellular growth. Together, these data define a dynamic interplay between the host tRNA landscape and viral infection and illuminate mechanisms governing host–virus interactions. sgRNA-seq of HFF with tRNA knockout CRISPR library, before and after 3 days of competition and of HCMV-infected HFF with tRNA knockout CRISPR library.Infected cells were FACS-sorted according to HCMV infection progression.