Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle
In clonal cultures, not all cells are equally susceptible to virus infection. Underlying mechanisms of infection variability are poorly understood. Here, we developed image-based single cell measurements to scrutinize the heterogeneity of adenovirus (AdV) infection. AdV delivers, transcribes and replicates a linear double-stranded DNA genome in the nucleus. We measured the abundance of viral transcripts by single-molecule RNA fluorescence in situ hybridization (FISH), and the incoming ethynyl-deoxy-cytidine (EdC)-tagged viral genome by copper(I)-catalyzed azide-alkyne cycloaddition (click) reaction. The early transcripts increased from 2-12 hours, the late ones from 12-23 hours post infection (pi), indicating distinct accumulation kinetics. Surprisingly, the expression of the immediate early transactivator gene E1A only moderately correlated with the number of viral genomes in the cell nucleus, although the incoming viral DNA remained largely intact until 7 hours pi. Genome-to-genome heterogeneity was found at the level of viral transcription, as indicated by colocalization with the large intron containing early region E4 transcripts, uncorrelated to the multiplicity of incoming genomes in the nucleus. In accordance, individual genomes exhibited heterogeneous replication activity, as shown by single-strand DNA-FISH and immunocytochemistry. These results indicate that the variability in viral gene expression and replication are not due to defective genomes but due to host cell heterogeneity. By analyzing the cell cycle state, we found that G1 cells exhibited the highest E1A expression, and significantly increased the correlation between E1A expression and viral genome copy numbers. This combined image-based single molecule procedure is ideally suited to explore the cell-to-cell variability in viral infection, including transcriptional activators and repressors, RNA splicing mechanisms, and the impact of the 3-dimensional nuclear topology on gene regulation. Author SummaryAdenoviruses (AdV) are ubiquitous pathogens in vertebrates. They persist in infected people, and cause unpredictable outbreaks, morbidity and mortality across the globe. Here we report that the common human AdV type C5 (AdV-C5) gives rise to considerable infection variability at the level of single cells in culture, and that a major underlying reason is the cell-to-cell heterogeneity. By combining sensitive single molecule in situ technology for detecting the incoming viral DNA and newly synthesized viral transcripts we show that viral gene expression is heterogeneous between infected human cells, as well as individual genomes. We report a moderate correlation between the number of viral genomes in the nucleus and immediate early E1A transcripts. This correlation is increased in the G1 phase of the cell cycle, where the E1A transcripts were found to be more abundant than in any other cell cycle phase. Our results demonstrate the importance of cell-to-cell variability measurements for understanding transcription and replication in viral infections.