Coupling technologies reveal viral gene defects in single cells

From the Bloom lab, Basic Sciences Division

The innate immune system is the body’s first line of defense and can be activated immediately once a pathogen attacks. The defense mechanisms provided by the innate immune system are a formidable barrier to the influenza virus. One method of protection is to recognize the viral RNA present within infected cells as foreign, which leads to the secretion of interferons (IFN) that stimulate expression of hundreds of antiviral, interferon-stimulated genes (ISG). Nevertheless, infection is extremely heterogeneous within cells; the influenza virus rarely triggers IFN production by infected cells, and it is not understood why only some infected cells trigger innate immune responses. The influenza virus is capable of great genetic diversity, and is known to evade IFN induction. However, due to its high mutation rate, individual virions often harbor genetic defects that could impair these immune evasion strategies. Identifying mutations present in each virion would be crucial to determine how viral genetic diversity influences cellular heterogeneity during infection, yet there is a lack of existing technologies to address this need. 

Dr. Jesse Bloom from the Basic Sciences division, and his laboratory, along with collaborators from the University of Cambridge developed a new technique to do just that.  Dr. Bloom explained: “It's long been known that influenza viruses have a high mutation rate, but up until our work it had been impossible to identify the specific mutations present in any given virion. We developed a new technique to determine the sequence of the virions infecting individual cells.” This work was published in a recent issue of Journal of Virology.

Led by Dr. Alistair Russell, a former postdoc in the Bloom lab who is starting his laboratory at UCSD this August, the authors coupled single-cell transcriptomics with long-read PacBio sequencing of viral genes in the first study to directly observe the full spectrum of these mutations across single cells. “This method was inspired by the observation that standard single-cell techniques only measure the expression of genes, but that long-read PacBio sequencing would enable us to also determine the sequences of the viral genes,” Dr. Bloom said. 

Approach for combined single-cell transcriptomics and viral long-read sequencing of single influenza virus-infected cells that express IFN.
Approach for combined single-cell transcriptomics and viral long-read sequencing of single influenza virus-infected cells that express IFN. Figure from publication

The authors first developed a system to identify and enrich rare IFN+ cells by creating cells that carried IFN reporters.  These cells were infected with barcoded viruses, following which mRNA from individual cells was converted to cDNA and tagged with cell-specific barcodes.  Cellular transcriptomes were quantified by standard single-cell sequencing, while viral genes were sequenced by PacBio. This approach generated both expression of each viral gene in every individual cell, as well as the complete sequences of viral genes in infected cells.  As such, the virus genotype in addition to the abundance of viral components can be measured for the first time.

From this, the authors identified four types of defects that increased IFN induction and carried out a series of experiments to further validate their findings. “Our work ended up shedding light on an important question: how does the innate immune system identify that we've been infected with a virus? We showed that several different types of mutations in the virus make the cells more likely to detect that they've been infected,” Dr. Bloom explained. 

Viral genotypes and infection outcomes in single cells.
Viral genotypes and infection outcomes in single cells. Figure from Dr. Bloom

This work presents an unbiased survey of the breadth of viral variation in individual infected cells, and shows that IFN induction cannot be determined by a single type of viral defect. Innate immune detection of influenza is thus a multifaceted process that cannot be ascribed to a single viral genetic cause; viral genetic defects also do not fully explain the heterogeneity among influenza virus-infected cells.  There is much to investigate, and this is just the beginning. “We hope that this technique will help to address many other outstanding questions in virology, as we now know it is possible to measure how naturally occurring mutations in viral populations impact their interactions with host cells,” Dr. Russell said.  

Russell AB, Elshina E, Kowalsky JR, te Velthuis AJW, Bloom JD. 2019. Single-cell virus sequencing of influenza infections that trigger innate immunity. J Virol 93:e00500-19.

This work was supported by the National Institutes of Health, Burroughs Wellcome Fund, Damon Runyon Cancer Research Foundation, Washington Research Foundation, University of Washington, Wellcome Trust and Royal Society, Isaac Newton Trust, the University of Cambridge and the Howard Hughes Medical Institute.