Researchers develop a defective synthetic SARS-CoV-2 genome that inhibits viral replication

A shorter genome comprising only part of the SARS-CoV-2 RNA genome competes with the normal virus for growth and can potentially prevent viral replication.

The devastating impact of the 2019 coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to significant efforts to curb its transmission. When RNA viruses, such as SARS-CoV-2, replicate, there may be versions of the virus that have large deletions in the viral genome, making the virus defective. These defective genomes can still replicate in the presence of the full-length virus. Because they are shorter, they can replicate faster than the original virus and compete with the integral virus for replication, stopping the virus from growing and spreading.

3D rendering of SARS-CoV-2 microbe with RNA molecule inside. Image Credit: Vchal / Shutterstock

These defective interfering genomes (DI) are common in coronaviruses, but DI has not yet been reported for SARS-CoV-2. It is generally believed that DI particles are the result of inefficient replication or that they have regulatory functions. Researchers from Pennsylvania State University, USA, developed a synthetic DI construct for SARS-CoV-2 and reported their results in a new study published in bioRxiv * prepress server.

A synthetic defective interfering genome

They prepared synthetic DI genomes from parts of the SARS-CoV-2 genome and tested whether DI genomes could replicate in cells infected with both the synthetic genome and wild type (WT) virus.

They made their construct using three portions of the virus genome: the 5 ‘UTR part and the 5’ part of the non-structural protein 1 (nsp1) in ORF1a, a part of nsp15 with the packaging signal and the 3 ‘part sequence. of the sequence N, ORF10 and 3’UTR. The synthetic DI genome was 2,882 nucleotides (nt) long, approximately 9.6% of the WT virus genome length. In addition, the authors also prepared a second shorter synthetic genome approximately 800 nt long without the packaging portion.

The team prepared the two genomes as DNA and inserted them into the plasmids, transcribing them in vitro to make the ANNs. The prepared RNAs were inserted into SARS-CoV-2 infected Vero-E6 cells.

Because the synthetic genomes of DI degrade rapidly and do not replicate well, the authors were unable to study how they replicated. However, the DI genome with the three portions reduced SARS-CoV-2 replication by approximately half, 24 hours after transfection. The shorter DI genome had no effect on the growth of SARS-CoV-2.

DI reduces the amount of SARS-CoV-2 by half; replicates 3 times faster; and it is transmitted with the same efficiency. Yellow: DI in coinfections; blue: WT in coinfections; gray: WT in infections without DI. a: growth rates (absolute amount relative to the 4-hour quantity) of WT in controls and co-infections; growth compared to controls at the same time; and details at 24 hours. b: 24 hours after infection the supernatant was used to infect new cells. Transmission efficiency is the quantity measured by qRT-PCR immediately before the passage divided by the average quantity measured almost immediately (4 hours) after the passage. c: growth rates (absolute amount relative to the 4-hour quantity) of WT in controls and co-infections; growth compared to controls at the same time; and details at 24 hours. Growth rates (absolute amount relative to the 4-hour amount) of WT and DI in co-infections; growth relative to that of WT in co-infections at the same time; and details at 24 hours.

One day after the transfection, the team infected new cells using the supernatant. Both the WT virus and the DI genome were seen starting four hours after infection, but they did not see the DI genome without the packaging portion. There was no difference in the transmission rate of the DI genome and the WT virus, suggesting that the shorter DI genome is as infectious as the WT virus.

In samples infected with both the DI genome and the normal virus, the number of regular virions decreased by half in 24 hours. The DI genome grew about three times faster than the WT virus. The results suggest that even a small amount of the DI genome can strongly influence WT virus replication.

The defective genome competes with the normal virus as it grows

The authors also modeled the dynamics of the system to understand intra-cellular competition between the DI genome and the normal virus. Similar to predictions from previous models, the DI genome and the WT virus coexist if the replication advantage of the DI genome is below a critical threshold. Above that threshold, the DI genomes will kill all WT viruses. This threshold depends on the number of genomes within the range of the viral protein produced by the normal virus.

Therefore, the model suggests that the DI genome will increase over time and kill the WT virus genome. However, because the authors measured only five passages, in which they saw an increase in the DI / WT genome ratio, they write that they were unable to verify whether this early reduction would lead to the extinction of the WT virus.

“Interference with the WT virus is the most notable effect of our DI construct,” the authors write. The DI particles serve no purpose for the WT virus; rather, they exist as parasites of the normal virus. Because they can replicate faster than the full-length virus when co-infected with the virus, they are suitable as potential antivirals.

As replication proceeds, the DI genome continues to increase in frequency and the process becomes more effective, ultimately leading to the death of both the normal virus and the DI genome. Although DI constructs have potential as antivirals, they haven’t been explored much. HIV and influenza viruses are not ideal candidates for this approach as they have short genomes and complex life cycles. However, coronaviruses are ideal for this approach as they have a long single-stranded RNA genome and simple life cycles and could be further explored for SARS-CoV-2.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behaviors, or treated as consolidated information.