Coronavirus biology: viral RNA synthesis, origin, and vaccines

In a recent study published in Current Research in Immunology, researchers analyzed the characteristics of coronaviruses (CoVs) and novel CoV vaccine candidates.

Study: Nature of viruses and pandemics: Coronaviruses. Image Credit: 3DJustincase/Shutterstock
Study: Nature of viruses and pandemics: Coronaviruses. Image Credit: 3DJustincase/Shutterstock

Background

CoVs are a group of positive-strand, enveloped ribonucleic acid (RNA) viruses belonging to the Nidovirales order. There are four genera within the Orthocoronavirinae subfamily: BetaAlphaDelta, and GammaCoV. These four genera of viruses can infect numerous birds and mammals, even humans, and cause various clinical signs according to the affected organ and tissue.

Seven known CoVs can infect humans. Among these CoVs, HCoV-OC43, HCoV-229E, HCoV-HUK1, and HCoV-NL63 produce mild infections comparable to common colds. The Middle East respiratory syndrome coronavirus (MERS-CoV), the severe acute respiratory syndrome coronavirus (SARS-CoV), and the SARS-CoV-2, on the other hand, produce potentially lethal severe respiratory infections. Moreover, the death rates and transmissibility of the three significantly pathogenic human CoVs vary.   

About the study 

In the current review, the researchers summarized the key CoV biology concepts that affect the establishment of antiviral approaches, like viral RNA synthesis and source. In addition, they reviewed new CoV vaccine candidate methods based on RNA replicons generated from CoV.

Replication, transcription of CoVs, and their genome's stability

The CoV polyproteins like polyprotein 1a (pp1a) are co- and post-translationally converted into 16 nonstructural proteins (nsps). Besides, most CoV nsps are responsible for subgenomic messenger RNA (sgmRNA) production and viral genome replication. While CoV genome replication necessitates continuous RNA synthesis, transcription was a discontinuous procedure during sgmRNAs synthesis, which is peculiar among RNA viruses.

Transcription comprises a template-switching during the subgenomic negative-strand RNA synthesis to incorporate a replica of the leader sequence into the developing negative RNA. Factors such as high-order RNA-RNA contacts and viral and cell protein-RNA adhesion play a role in the regulation of CoV transcription.

Additionally, the CoV RNA genome's variability is increased significantly by the discontinuous synthesis. Furthermore, copy-choice RNA recombination with high frequency and similarity assistance were analogous to CoV transcription.

Origin of human CoVs

The three fatal CoVs that affect humans have zoonotic origins, including the SARS-CoV-2. These CoVs have bats as their natural reservoir, and an intermediary host transmits the virus to humans. Humans have contracted the MERS-CoV from camels and SARS-CoV from civet cats. Further, SARS-CoV-2 was most probably transmitted from raccoons to humans since a virus with a 99.998% sequence similarity to SARS-CoV-2 was discovered in the raccoon excrement found in the metal cages of the Huanan market, Wuhan. The threat of the emergence of novel pathogenic variants increases when the animal-adapted SARS-CoV-2 can infect humans and traverse species barriers, as it did with minks.

MERS-CoV vaccine candidate based on RNA replicon

The authors' laboratory has previously developed CoV-derived propagation-defective and replication-competent RNA replicons as potential platforms for vaccine development. Compared to traditional messenger RNA (mRNA)-based vaccinations, these replicons used an RNA dose that was 60 times lower.

The first method for engineering CoV genomes was created by cloning a copy of the CoV complementary deoxyribonucleic acid (cDNA) into bacterial artificial chromosomes (BACs). This method has proved crucial for generating self-replicating RNAs from CoV RNA genomes by deleting a group of genes.

The team found that in the absence of the complementation of the envelope (E) protein, virus-like particles (VLPs) carrying recombinant MERS-CoV-ΔE (rMERS-CoV-ΔE) RNA replicons were not contagious. These VLPs were artificially liberated from infected cells through freeze-thawing and stayed non-infectious, confirming their safety. A single rMERS-CoV-ΔE replicon immunization safeguarded the highly vulnerable keratin 18-human dipeptidyl peptidase 4 (K18-hDPP4) transgenic mice from a deadly viral infection.

Deleting about five separate genes in a mouse-adapted virus context (rMERS-MA30-Δ[4a, 3, 4b, E, 5]) improved the safety of the RNA replicon based on rMERS-CoV-ΔE. The likelihood of recombination with other human CoVs, like HCoV-229-E, was decreased by deleting other non-essential genes in the RNA, such as 5 and 3. The team discovered that in hDPP4-knock-in (KI) mice, the rMERS-MA30-Δ[4a, 3, 4b, E, 5] replicon caused immunity against a lethal MERS-MA30 virus dose and advanced sterilizing immunity with high neutralizing antibody levels.

Next generation SARS-CoV-2 vaccine candidates based on safe and efficient propagation-defective replication-competent RNA replicons

The authors sought to totally or partially delete non-replicase and replicase genes located at distal genome domains to boost the biosafety of the SARS-CoV-2-stemmed replicon to avoid the possibility that a single recombination incident with circulating CoVs could reestablish virus virulence and the potential of dissemination. The generation of VLPs from packaging cell lines trans-complementing RNA replicons with the proteins necessary for replicon propagation was the most effective method for producing a SARS-CoV-2-stemmed RNA replicon.

SARS-CoV-2 VLPs offered complete protection from virus infections, as demonstrated by the evaluation of the SARS-CoV-2-stemmed RNA replicon vaccine option in humanized K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice immunized intranasally. To further verify the safety and efficacy of the RNA replicon vaccine candidate, the scientists assessed it in non-human primate animals and hamster models.

Conclusions

Overall, in the present manuscript, the authors stated that CoVs possess the broadest genomes across RNA viruses and can store a significant amount of data without integrating their genomes while replicating in the cell cytoplasm. The transcription of the CoV sgmRNAs, which involves a template switch and mimics a high-frequency recombination strategy that may promote the virus genome variability, was a discontinuous procedure in contrast to continuous virus replication.

The three lethal human CoVs, MERS-CoV, SARS-CoV-2, and SARS-CoV, originated through zoonotic transmission. The spike (S) protein of SARS-CoV-2 contains a furine proteolytic area facilitating the virus activation in any tissue, enabling this CoV strain to be substantially pathogenic and polytropic.

By eliminating the E protein gene required for virulence and vital viral morphogenesis and accessory genes 4a, 3, 5, and 4b accountable for innate immunity antagonism, a propagation-deficient RNA replicon was synthesized utilizing MERS-CoV as a template to weaken MERS-CoV-Δ[4a, 3, 5, 4b, E]. This RNA replicon was a viable vaccine prospect for this virus and an intriguing platform for vector-based vaccine generation because it was strongly damped and induced sterilizing immunity following a single immunization via the intranasal route in transgenic mice harboring the MERS-CoV receptor.

Moreover, the team noted the possibility of developing an approach for designing RNA replicon vaccines targeting other CoVs pathogenic to humans.

Journal reference:
Shanet Susan Alex

Written by

Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.

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