Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, with most infected cells giving rise to no or few viral progeny while some cells produce thousands. Analysis of Herpes Simplex virus 1 (HSV-1) infection by population-averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that these cells cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of β-catenin to the host nucleus and viral replication compartments, and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

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9Viral infection is usually studied at the population level by averaging over millions of cells. However, 10 infection at the single-cell level is highly heterogeneous. Here, we combine live-cell imaging and single-11 cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of 12 primary human cells. We find extreme variability in the level of viral gene expression among individually 13 infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-14 viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo 15 cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the 16 recruitment of beta-catenin to the host nucleus and viral replication compartments and is required for late 17 viral gene expression and progeny production. These findings uncover the transcriptional differences in 18 cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-19 1. 20 21 24 throughout the host life with occasional reactivation. Here, we focus on the lytic part of the virus life cycle. 57While lytic infection is usually asymptomatic, in some cases -particularly in immune-compromised 58 individuals and infants -it can results in life threatening conditions such as meningitis and encephalitis. 59To initiate infection, HSV-1 must bind to its receptors, enter the cytoplasm, travel to the nuclear pore and 60 inject its linear double-stranded DNA into the host nucleus (Kobiler et al., 2012). Once in the nucleus, viral 61 gene expression proceeds in a temporal cascade of three classes of viral genes: immediate-early, early and 62 late Roizman, 1974, 1975;Harkness et al., 2014). DNA replication occurs in sub-nuclear 63 structures, called replication compartments (RCs), that aggregate the seven essential replication proteins as 64 well as other viral and host proteins (de Bruyn Kops and Knipe, 1988;Liptak et al., 1996; Weller and Coen, 65 2012;Dembowski and DeLuca, 2015;Dembowski et al., 2017; Reyes et al., 2017; Dembowski and DeLuca, 66 2018). ICP4 is the major viral trans-activator and is required for viral infection to progress beyond the point 67 of immediate-early gene expression. Upon viral DNA replication, ICP4 is predominantly localized in the 68 RCs, with some diffuse nuclear and cytoplasmic localization (Knipe et al., 1987;Zhu and Schaffer, 1995). 69Several studies applied high-throughput technologies to analyze the cellular response to HSV-1 infection 70 at the population level. RNA sequencing revealed a widespread deregulation of host transcription, including 71

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.

Detection of protein expression by MRI requires a high payload of Gd(III) per protein binding event. Presented here is a targeted AuDNA nanoparticle capable of delivering several hundred Gd(III) chelates to the HaloTag reporter protein. Incubating this particle with HaloTag-expressing cells produced a 9.4 contrast-to-noise ratio compared to non-expressing cells.