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Functional Single-Cell Genomics of Human Cytomegalovirus Infection

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Functional Single-Cell Genomics of Human Cytomegalovirus Infection bioRxiv preprint doi: https://doi.org/10.1101/775080; this version posted October 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Functional single-cell genomics of human cytomegalovirus infection Marco Y. Hein1, and Jonathan S. Weissman1, 1Department of Cellular and Molecular Pharmacology & Howard Hughes Medical Institute, University of California San Francisco, San Francisco CA 94143, USA Understanding how host factors and hundreds of viral genes or- rectly and their function through errors introduced by the host chestrate the complex life cycle of herpesviruses represents a DNA repair machinery (8). Cleavage of the viral DNA in fundamental problem in virology. Here, we use CRISPR/Cas9- non-essential regions has a moderate impact on genes proxi- based screening to scan at high-resolution for functional ele- mal to the cut site, but minimal impact on HCMV replication ments in the genome of human cytomegalovirus (HCMV), and and host cell viability, likely because DNA repair is fast rel- to generate a genome-wide mapping of host dependency and re- ative to the kinetics of replication ((8) and our data below). striction factors. Our data reveal an architecture of functional Thus, Cas9 represents an effective tool for making targeted modules in the HCMV genome, and host factor pathways in- volved in virus adhesion and entry, membrane trafficking, and disruptions in the viral genome. To enable high-resolution innate immune response. Single-cell analysis shows that the scanning of viral elements for a comprehensive functional an- large majority of cells follow a stereotypical trajectory in viral notation of the HCMV genome, we designed a single-guide gene expression space. Perturbation of host factors does not al- RNA (sgRNA) library that targets every protospacer-adjacent ter this trajectory, but can accelerate or stall progression. Con- motif (PAM) for S. pyogenes Cas9 (NGG PAM sequence versely, perturbation of viral factors creates discrete alternate present roughly every 8 bp) along the genome of the clinical ‘dead-end’ trajectories. Our results reveal a fundamental di- HCMV strain Merlin (Fig. 1A, Table S1). We delivered the chotomy between the roles of host and viral factors in orches- library into primary human foreskin fibroblasts engineered to trating viral replication and more generally provide a road map express Cas9, so that upon HCMV infection, each cell exe- for high-resolution dissection of host-pathogen interactions. cutes a cut at a defined position along the viral genome, col- Correspondence: [email protected], [email protected] lectively tiling its entirety. The betaherpesvirus HCMV is a pervasive pathogen that es- We mapped the phenotypic landscape by quantifying the tablishes lifelong infection in the majority of the human pop- abundances of individual sgRNA cassettes in cells surviving ulation. Activation of its lytic cycle triggers a characteristic infection relative to the initial population by deep sequenc- cascade of events, starting with stereotypical waves of viral ing (Fig. S1). We found that cutting phenotypes are rel- gene expression, continuing with the replication of the large, atively constant within individual genes, i.e. the determin- 235 kb dsDNA genome, and culminating in the budding of ing factor is which gene is targeted by Cas9, more so than newly assembled virions. A number of systematic studies the relative position of the target site within the gene. Ad- have described these phenomena on the level of the transcrip- jacent sets of genes also frequently had similar phenotypes. tome, the set of translated messages, and the proteome in time However, some gene boundaries were marked by abrupt phe- and space (1–6). These studies have highlighted the com- notype changes, arguing that here, strong functional conse- plexity of the process and have raised the question of how quences of Cas9-induced double-strand breaks are limited to hundreds of viral genes cooperate to manipulate the host and the immediate vicinity of the cut sites (Figs. 1B, S2, Table undermine its defense machinery. CRISPR technology pro- S1). vides us with tools to systematically measure the functional At a larger scale, changes in the direction and magnitude of contribution of each viral gene and to identify the host fac- the phenotypes defined six major genomic modules: Cuts in tors involved in productive infection (7). Here, we present both distal regions of the genome, which lack genes essen- systematic screens for both host and viral factors affecting tial for viral replication (9, 10), had minimal impact on host HCMV infection in primary human fibroblasts. To capture cell survival. As expected, targeting the two regions cover- the complexity of the molecular events during infection, we ing UL48A–UL73 and UL96–UL150, both of which contain recorded the transcriptomes of tens of thousands of single essential genes involved in viral DNA replication, packaging cells, monitoring how perturbation of critical host and viral and nuclear egress (9, 11), strongly protected infected cells factors alters the timing, course, and progression of infec- from death. Surprisingly, in the two remaining regions of the tion. Our data paint an unprecedented picture of the HCMV genome, we found that disruption of genes required for viral life cycle and its vulnerabilities to antiviral intervention. replication did not necessarily protect the host from death. Cuts within the UL32–UL47 region, which contains essen- High resolution functional landscape of the HCMV tial genes, led to a strongly increased ability of the virus to genome kill cells. The most strongly sensitizing phenotypes mapped It was recently shown that targeting individual essential her- to the known viral apoptosis inhibitors UL36, UL37, and pesvirus genes by CRISPR/Cas9 disrupts their expression di- UL38 (12). While this behavior can be rationalized for virally Hein & Weissman | bioRχiv | October 2, 2019 | 1–22 bioRxiv preprint doi: https://doi.org/10.1101/775080; this version posted October 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A (d)Cas9-expressing primary fibroblasts HCMV HCMV genome population surviving tiling library HCMV infection deep or sequencing human genome- lentiviral wide gene 1 gene 2 gene n packaging t0 population library uninfected population B 2 disruption ) 0 enhances 1 host survival 0 50,000 100,000 150,000 200,000 kb (surviving/t -12 disruption UL97 UL98 UL102 UL105 diminishes UL95 UL99 RNA5.0 log UL104 -2 host survival UL, distal UL32–UL47 UL48A–UL73 UL75–UL88 UL96–UL150 US, distal C ) 0 PDGFRA heparan sulfate Fig. 1. Virus and host-directed CRISPR screens map interferon response the phenotypic landscape of HCMV and its host’s depen- host restriction factors dency and restriction factors. (A) Experimental design (uninfected/t 2 NEDDylation for pooled, virus and host-directed CRISPR screening. log Cullin-RING Our HCMV tiling library or genome-wide human sgRNA li- COP9 signalosome braries were lentivirally delivered into primary human fore- apoptosis skin fibroblasts expressing the CRISPRi or CRISPRn ma- UNC50 chinery, followed by infection with HCMV. sgRNA cas- diminished enhanced RIC1/RGP1/RAB6A settes were quantified by deep sequencing in the initial host survival host survival COG complex (t0) population, the surviving population, and the unin- BORC complex fected control population. (B) Phenotypic landscape of the TRAPP complex III HCMV genome obtained by locally averaging the pheno- ERAD types of individual sgRNAs. Strong changes in the mag- SRP/translocon nitude of the phenotype coincide with gene-gene bound- ER stress aries (inset). (C) Results of host-directed CRISPRi screen essential Ragulator displayed as a scatter plot of average gene essentiality host genes NuA4 HAT complex (i.e. infection-independent phenotype; y-axis) vs. pro- log2(surviving/t0) tection/sensitization to death upon HCMV infection (i.e. infection-dependent phenotype; x-axis). encoded anti-apoptotic proteins, it extended to many other the initial population, as well as in an uninfected control pop- virus-essential genes without known anti-apoptotic roles, in- ulation to account for host gene essentiality (Fig. 1C, Table cluding the DNA polymerase processivity factor UL44. Sim- S2). ilarly, cuts in the central region spanning UL75–UL88 led to Our screen revealed a range of diverse host genes required slightly enhanced host cell death upon infection. Many genes for multiple steps in the viral life cycle. Genes involved in in this region are encoding essential structural components of the biosynthesis of heparan sulfate were among the strongest the viral envelope, tegument, and capsid. protective hits. Heparan sulfate proteoglycans on the cell Targeting essential viral genes, by definition, undermines the surface enable the adhesion of HCMV prior to cell entry production of viral offspring. The outcome for the host, how- (15, 16). Additionally, we found a range of vesicle trafficking ever, is more nuanced and sometimes counterintuitive. It ap- factors: RAB6A and its GEFs RIC1/KIAA1432 and RGP1, pears that disrupting essential genes involved in viral DNA the conserved oligomeric Golgi (COG) complex, members replication mostly protects the host. However, interfering of TRAPP complex III, and UNC50. These factors converge with the later steps of assembling new virions may not only on the Golgi apparatus and mediate both retrograde and an- be ineffective for protecting the host, but even place an addi- terograde transport, implying that they act downstream of vi- tional burden, leading to enhanced host cell death. ral entry. Some had previously been implicated in the in- ternalization of diverse bacterial and plant toxins, suggest- Genome-wide screen for host factors of HCMV infec- ing that HCMV and toxins exploit similar pathways for cell tion entry (13, 17–21).
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