NEW YORK – Using a pooled CRISPR interference and nuclease screening method, researchers have defined the contribution of each viral and host factor to human cytomegalovirus infection in primary human fibroblasts.
In a paper published on Monday in Nature Biotechnology, University of California, San Francisco researchers Marco Hein and Jonathan Weissman noted that understanding how viral and host factors interact and how perturbations affect infection is the basis for designing antiviral interventions. "Our results reveal the roles of host and viral factors and provide a roadmap for the dissection of host-pathogen interactions," they wrote.
Their team used Perturb-seq, a scalable approach that combines CRISPR-based genetic perturbations with rich phenotypic profiling by single-cell transcriptomics, to determine how genetic perturbation of critical host and viral factors alters the timing, course, and progression of infection. They recorded the transcriptomes of tens of thousands of CRISPR-modified single cells and found that most cells normally follow a stereotypical transcriptional trajectory. While perturbing critical host factors does not change the stereotypical transcriptional trajectory, it can stall, delay, or accelerate progression along the trajectory, allowing for precise pinpointing of the stage of infection at which host factors act.
Conversely, the researchers also found that perturbation of viral factors can create distinct, abortive trajectories.
First, they conducted systematic pooled CRISPR screens for both host and viral factors that affected the survival of primary human fibroblasts upon infection with human cytomegalovirus. By doing this, they were able to map the functional landscape of the cytomegalovirus genome by quantifying the abundance of individual single guide RNA, or sgRNA, cassettes in a population before and after infection.
Targeting essential viral genes undermined the production of viral offspring but didn't necessarily protect the infected cell, underscoring that successful viral replication and death of the infected cell are distinct phenomena. It also appeared that disrupting essential genes involved in viral DNA replication mostly protected the host, the researchers said.
However, they also found that interfering with the later steps of assembling new virions might not only be ineffective in protecting the host but might even place an additional burden.
The investigators also conducted a series of Perturb-seq experiments exploring the effect of targeting host and viral factors on the viral life cycle. Among their observations, they noted that targeting a viral factor didn't just stall infection at the stage where that factor became essential, as was the case with host factors. Rather, cells with virus-targeting guides assumed distinct areas in transcriptional space but only in infected cells.
These findings prompted them to examine the patterns of viral gene expression in infected cells. They found that the course of infection can be visualized as a trajectory by a rolling average of the positions of cells with increasing viral load. Cells with host-targeting sgRNAs all followed trajectories that were nearly congruent with the default trajectory (as defined by cells with control sgRNAs).
In marked contrast, cells with virus-targeting sgRNAs followed trajectories that diverged from the default. These differences were driven by viral expression patterns and not by concomitant variations in host gene expression.
"Based on our high-dimensional dataset, we organized host factors both by the transcriptional responses in cells where these factors are targeted and by how infection progresses in those cells," the authors wrote. "This provides a systematic classification of host factors by functional category that reveals a range of factors acting in viral entry, in early-stage infection, and as restriction factors."
They further noted that it would be interesting to investigate whether the dichotomy of host-directed versus virus-directed perturbations is a general feature across cell types in the case of cytomegalovirus and of other virus-host systems more generally, and added that this work could be used to inform the design of attenuated viral strains for vaccine development purposes, or serve as a blueprint for defining the vulnerabilities of other viruses to genetic or pharmacological interventions.