Talking a lot but saying nothing: no link between viral transcription and progeny production

From the Bloom Lab, Basic Sciences and Public Health Sciences Divisions.

As humans, we’re often obsessed with proving ourselves unique or irreplaceable in some way – we crave individuality. “Be Yourself!” we proclaim, “because an original is worth more than a copy.” However, in the world of viruses Copies are King. “Success” to a virus means duplicating itself over and over, creating as many copies as possible in order to infect more hosts and ensure survival of the viral lineage. Dr. Jesse Bloom, a Professor in the Basic Sciences and Public Health Sciences Divisions at Fred Hutch, studies how naturally occurring mutations in these fast-copying viruses influence their ability to infect hosts. However, David Bacsik, a graduate student in the Bloom Lab, had an even more basic question: how many copies, or progeny, are produced during individual infection events? And does this have any relation to how transcriptionally active a given virus is during infection? The results of his study were recently posted on bioRxiv.

“Looking at cells infected with influenza virus, we wanted to understand how many viral genes each cell transcribes and how many new virions each cell generates,” explained Bacsik. “What's the relationship between these two processes? It's a pretty basic question about how influenza virus functions, but until very recently, it couldn't be examined empirically.” The Bloom Lab specializes in harnessing highly detailed datasets to answer fundamental virology questions, and this study is no different. David analyzed data from single cells infected with influenza, quantifying both the number of progeny each infected cell produced, and the amount of viral mRNA being made, and looked for a correlation between the two. Several viral gene products are crucial for putting together the packaging that allows new virus particles to leave cells and find new targets. Therefore, you might not expect viable, infectious progeny from cells that lacked transcription of those genes (or that expressed mutated forms). “The naive model I had imagined was one where making more viral transcripts led directly to making more viral progeny,” explained Bacsik. “But the data didn't support this at all. The most exciting moment was realizing that influenza virus transcription is not strongly correlated with progeny production in single cells. In [fact], the cells that transcribe the most viral mRNA are not the cells producing the most progeny virions.” The authors proposed a partial explanation for this unexpected result: transcription of the influenza NS gene. Infected cells that fail to express the NS gene make very few progeny, and one of the proteins encoded by the NS gene regulates the switch from transcription to genome replication, another crucial step in assembling new virus particles. However, the authors point out that “absence of NS and other viral genetic defects only explain part of the discordance between viral transcription and progeny production in single cells, since these two properties are often discordant even in cells expressing unmutated copies of all viral genes.”

A graphic showing the technical details of the screen that David Bacsik and Dr. Jesse Bloom used to interrogate how viral replication and viral transcription are related.
In this study, Bacsik et al used a barcoded library of viruses to simultaneously measure viral transcription and production of viral progeny. Figure provided by David Bacsik.

Moving forward, Bacsik says that the team wants to build on this study in two ways. “First, we'd like to scale up so that we have the power needed to start exploring how host cell state affects progeny production,” he said. “Second, we'd like to perform similar experiments in differentiated lung model systems, so that we can measure progeny production directly from a variety of lung cell types.” Dr. Bloom chimed in, “the unique thing about this study is that we manage to measure the most relevant outcome of viral infection---how many new virions are made---at the level of single cells. It’s interesting that this outcome doesn’t correlate very well with some other measures such as viral transcription, showing that many infections are aberrant at the single-cell level.” Clarifying the relationship between viral transcription and production of viral progeny could have implications for the way we think about viral infections in the clinic, and even how we treat infection. For instance, viral infections usually involve several cell types within a tissue – what if the production of infectious viral particles is correlated more with cell type than with viral transcription? These results suggest that a high load of viral mRNA does not necessarily correlate with high infectivity for influenza, and that a huge amount of heterogeneity exists even within one infection within one cell type.

“Two things were hard about this project,” Bacsik confessed. “First, high quality sequencing data had to be generated from very small samples of cells, and even smaller samples of viral progeny; recovering as much material as possible at every step was a major focus of the bench work. Second, we generated three types of sequencing data that aren't often analyzed in concert. We had to develop a large computational pipeline to integrate all of the information we generated in ways that accounted for the strengths and limits of each technique we used.” Nonetheless, David’s persistence and innovation in analyzing these disparate data led to an excitedly unexpected result, and he is grateful for the Fred Hutch resources that supported him and his lab members. “The scientists in the cores at Fred Hutch have provided important guidance at several stages of this project. Specifically, I owe thanks to Andy, Marty, and Cassie Sather in the sequencing core and Andrew Berger in the flow cytometry core. Their expertise was incredibly helpful when I was developing this system and when I was optimizing three different high-throughput sequencing assays to work well with low input material.”

This work was supported by the National Institutes of Health, the National Institute of Allergy and Infectious Diseases, the Howard Hughes Medical Institute, and the Burroughs Wellcome Fund.

Bacsik DJ, Dadonaite B, Butler A, Greaney AJ, Heaton NS and Bloom JD. 2022. Influenza virus transcription and progeny production are poorly correlated in single cells. bioRxiv. doi: