Evolution of naturally arising SARS-CoV-2 defective interfering particles

Context

Defective interfering particles (DIPs) arise during virus propagation. They are particles containing degenerate forms of the virus genome, which interfere with the replication of the parental virus but do not replicate themselves. Historically, such particles were considered artefacts of virus propagation in the laboratory, however, studies have shown that defective viral genomes are present in patients infected with viruses such as Influenza A and Hepatitis C. There is much interest in developing anti-viral therapies based on DIPs.

Objectives

Here in a study led by Jerry Pelletier and Tom Hobman at McGill University and University of Alberta respectively, we characterize DI particles that arose following serial passaging of SARS-CoV-2 at high infection levels.

Methods

Investigators supported by CoVaRR-Net were able to use cutting-edge technologies to identify and directly analyze the complete sequence of DIP RNA molecules representing the DI genomes from a complex mixture of virus RNAs, by a method able to analyze individual, complete RNA molecules, called native RNA sequencing by Nanopore. The prominent DI RNAs identified were functionally characterized showing that they have lost ~84% of the SARS-CoV-2 genome and are able to suppress the original virus. The group produced synthetic forms of the defective virus’ RNA and found that it can be introduced into infected cells to interfere with virus production.  Importantly, the defective virus is only replicated when cells are infected and is inactive in uninfected cells. Furthermore, a novel, naturally occurring DIP RNA representing a fusion of the SARS-CoV-2 Nsp1 protein has been identified by direct RNA sequencing and a synthetic version of it, as well as of the corresponding protein, were shown to inhibit virus replication in infected cells

Significance and future research

The investigators have shown that defective interfering particles can cause cyclical changes in the amount of virus produced by infected cells, since they not only compete with but also rely on parental viruses for propagation. Through detailed analysis, the investigators detected a new fusion RNA molecule and a corresponding protein that alone can suppress viral replication.  Overall, these results identify naturally selected defective viral genomes that emerge and stably propagate in the presence of parental virus.  The next step is to develop therapeutic strategies to deliver the RNA encoding the DI particle by inhalation through the nose and test them extensively in mouse models.

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