Influenza virus transcription and progeny production are poorly correlated in single cells

The ultimate success of a viral infection at the cellular level is determined by the number of progeny virions produced. However, most single-cell studies of infection quantify the expression of viral transcripts and proteins, rather than the amount of progeny virions released from infected cells. Here we overcome this limitation by simultaneously measuring transcription and progeny production from single influenza-virus-infected cells by embedding nucleotide barcodes in the viral genome. We find that viral transcription and progeny production are poorly correlated in single cells. The cells that transcribe the most viral mRNA do not produce the most viral progeny, and often represent aberrant infections that fail to express the influenza NS gene. However, only some of the discrepancy between transcription and progeny production can be explained by viral gene absence or mutations: there is also a wide range of progeny production among cells infected by complete unmutated virions. Overall, our results show that viral transcription is a relatively poor predictor of an infected cell's contribution to the progeny population. Overall design: We performed single-cell RNA sequencing of influenza-infected cells. The influenza virus library we used contained nucleotide barcodes that linked the infected cell to the progeny it produced. We quantified the physical progeny produced by infected cells by sequencing viral barcodes from the supernatant. We quantified infectious progeny produced by sequencing viral barcodes from a new set of cells infected with the supernatant. We also sequenced the full-length viral transcripts in the single-cell cDNA library using long-read sequencing.