As coronavirus disease 2019 (COVID-19) continues to spread worldwide, the rapid development and rollout of vaccines against its causative pathogen – the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – has raised hopes for many worldwide that a return to ‘normality’ may be on the horizon.
New drugs have also been urgently sought to mitigate severe or critical COVID-19 and prevent death. This has been a particularly pressing concern in parts of the world where vaccine access has been alarmingly low.
What is the importance of this study?
The rise of new SARS-CoV-2 variants has been disturbing since some have exhibited heightened infectivity, reduced neutralization by vaccine- or prior infection-induced antibodies and possibly even increased virulence. A fascinating new preprint describes a proof of concept that novel virus-specific targets can be identified in order to provide a broadly effective pharmaceutical intervention.
SARS-CoV-2 is a virus with a positive-sense ribonucleic acid (RNA) genome.
The current study, which is available on the bioRxiv* preprint server, reports the use of a modified deoxyribonucleic acid (DNA) oligonucleotide that has a 3’ polyadenine (polyA) tail to bind to the 5’-polyU tract in the mouse coronavirus (MHV-A59). Treatment of mouse cells infected with MHV-A59 with this molecule led to a marked reduction in cell deaths.
This indicates a unique mechanism of action against this virus, promising in terms of the development of new drugs to inhibit coronavirus infection within host cells. This could probably be extended to SARS-CoV-2. Since the 5’-polyU tract is observed in all infected cells soon after viral entry, it could be a biomarker for the detection of infection in the early stages.
Targeting the polyU tract
The lack of vaccines has led to a widening of the gap between vaccine ‘haves’ and ‘have-nots’. Some countries have resorted to covering as much of their population as possible with a single dose of vaccines with two dose regimens and delaying the second dose while supplies are low. While this strategy could provide a wider net of partial immunity, it could also prompt the emergence of new immune-escape variants, often with higher transmissibility or pathogenicity.
Drugs targeting SARS-CoV-2 genomic coding regions may also be rendered ineffective by such mutations. A possible alternative could be to design or develop drugs that inactivate segments of the viral genome that are essential for viral replication. The development of resistance to such drugs by mutation would be near impossible, precluding the development of new escape variants under selection pressure from drugs or vaccines.
This type of strategy is therefore effective at preventing the development of escape variants. In the current study, the researchers examined the 5’ polyuridines (5’ poly-U) tract on the antigenome (minus RNA genome) of the positive-sense viral RNA. The 5’ polyU tract is necessary as a template for the synthesis of the positive-sense strand’s 3’-polyA tail and vice versa.
A mutation in this polyU tract would almost certainly block this synthesis, which in turn blocks the completion of the virus lifecycle by preventing the replication of the full-length viral genome. Earlier research has firmly established the need for the 5’-polyU tract on this antigenome strand for virus survival and infectivity among positive-sense RNA viruses.
Of great value is the added fact that host cells lack this 5’-polyU tract completely, meaning it is a virus-specific target. However, RNA polymerase III within eukaryotic cells does lead to the formation of 3’-polyU tract as part of the termination mechanism for the transcription of some small RNA molecules, and scientists are being careful to ensure that this is distinguished from viral 5’-polyU to prevent off-target drug effects.
How was the target sequence designed?
In the current study, this was ensured by using a DNA oligo with a complementary 3’-polydA tract. The oligo was designed to be resistant to degradation by nucleases, less toxic, but with high binding affinity to the complementary RNA strand. At the same time, avoiding the use of oligonucleotides tailored to the viral genes prevents the use of alternative polyA sites, especially because there is no cryptic polyA signal near the target sequence.
Oligos are large in size and carry a negative charge, besides being easily degraded by host nucleases. For the same reason, they are slow to enter cells, have a low half-life and may be immunogenic, all of which make them poor choices for drug development.
Recent advances have made it possible to create vehicles that carry oligos in complex form, thus neutralizing the charge and avoiding their degradation. Most often, these drug delivery vehicles are composed of tertiary or quaternary amines with a positive charge attached to a polymer or lipid.
By choosing the right vehicle and fine-tuning its structure, the synthetic ease, stability of the oligo and carrier, half-life, and inclusion of the targeting agent can be optimized. For instance, the use of lipids as delivery agents is supported by the current messenger ribonucleic acid (mRNA) COVID-19 vaccines.
In the current study, a polymer 1-based vehicle was used. An excess oligo concentration was provided to ensure that the Replication-Transcription complex (RTC) would be formed as expected in the cytoplasm for the virus-directed synthesis of its genomic and subgenomic RNAs.
The extra-high oligo levels were intended to compensate for the known possibility that viral replicase nonstructural protein 4 (nsp4) could cause structural defects in the double membrane vesicles (DMVs) formed by coronaviruses during viral replication. These are essential to anchor the RTC.
When used to treat fluorescent-tagged MHV-A59-infected mouse cells, the oligo inhibited cytopathic effects induced by the virus. The pre-exposure of the cells to the virus suggests that the inhibitory effect is not because viral entry is prevented.
Secondly, viral replication seems to continue despite oligo treatment, but cell death was reduced markedly relative to untreated cells.
What are the implications?
The current study indicates the utility of the oligo approach in preventing and delaying cell death following virus entry, despite the presence of viral replication. The effect of the oligonucleotide may thus be to reduce the synthesis of the fully formed virion. This needs to be confirmed by further studies on the infectivity of the virus following oligo treatment.
“Here we have presented preliminary evidence to establish the 5’-polyU tag as a legitimate target for the successful containment of virus spread.” The careful design of the oligo tag, and the polymer vehicle, contributed to its success, since the coronavirus is known to be in the cytoplasm, allowing for efficient binding between the oligo and the target.
The role of the 5’-polyU tract in the viral lifecycle is well known, and is essential for the synthesis of the polyA template of the virus. N-terminal RdRp-associated nucleotidyl-transferase activity may potentially allow this to be bypassed by adding polyA without a template. However, the RdRp acts to add the 3’-polyA tail efficiently only when the template 5’-polyU tract is present on the other strand, and this activity is conserved among coronaviruses.
This addition of the 3’-polyA tail stabilized and matured the immature positive-sense RNA strand. While oligo-5’-polyU binding could simulate a reduction in the length of the 5’-polyU stretch, leading to immune escape, it may preclude the production of fully formed viral particles. Further work will be required to uncover the mechanisms by which this occurs to cause cell death.
Experimental establishment of an indispensable target like this 5’poly-U tract on the antigenome of coronaviruses may assist in the development of effective drugs that prevent the generation of drug-escaping virus variants.”
Not only does blocking the 5’-polyU stretch disrupt a key replication mechanism of the virus, leading to the formation of a dead or non-functional virus, but the fact that this stretch appears soon after infection may allow it to be used to detect early infection.
This may overcome many of the issues encountered with the prevalently used real-time PCR- based detection systems, such as the lack of enough template early in the infection which may drive false negative results.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.