Evolution of the coronavirus spike protein in the full-length genome and defective viral genome under diverse selection pressures.

How coronaviruses evolve by altering the structures of their full-length genome and defective viral genome (DVG) under dynamic selection pressures has not been studied. In this study, we aimed to experimentally identify the dynamic evolutionary patterns of the S protein sequence in the full-length genome and DVG under diverse selection pressures, including persistence, innate immunity and antiviral drugs. The evolutionary features of the S protein sequence in the full-length genome and in the DVG under diverse selection pressures are as follows: (i) the number of nucleotide (nt) mutations does not necessarily increase with the number of selection pressures; (ii) certain types of selection pressure(s) can lead to specific nt mutations; (iii) the mutated nt sequence can be reverted to the wild-type nt sequence under the certain type of selection pressure(s); (iv) the DVG can also undergo mutations and evolve independently of the full-length genome; and (v) DVG species are regulated during evolution under diverse selection pressures. The various evolutionary patterns of the S protein sequence in the full-length genome and DVG identified in this study may contribute to coronaviral fitness under diverse selection pressures.

Unveiling the biology of defective viral genomes in vitro and in vivo: implications for gene expression and pathogenesis of coronavirus.

BACKGROUND: Defective viral genome (DVG) is a truncated version of the full-length virus genome identified in most RNA viruses during infection. The synthesis of DVGs in coronavirus has been suggested; however, the fundamental characteristics of coronavirus DVGs in gene expression and pathogenesis have not been systematically analyzed. METHODS: Nanopore direct RNA sequencing was used to investigate the characteristics of coronavirus DVGs in gene expression including reproducibility, abundance, species and genome structures for bovine coronavirus in cells, and for mouse hepatitis virus (MHV)-A59 (a mouse coronavirus) in cells and in mice. The MHV-A59 full-length genomic cDNAs (~ 31 kilobases) were in vitro constructed to experimentally validate the origin of coronavirus DVG. The synthesis of DVGs was also experimentally identified by RT-PCR followed by sequencing. In addition, the alterations of DVGs in amounts and species under different infection environments and selection pressures including the treatment of antiviral remdesivir and interferon were evaluated based on the banding patterns by RT-PCR. RESULTS: The results are as follows: (i) the structures of DVGs are with diversity, (ii) DVGs are overall synthesized with moderate (MHV-A59 in cells) to high (BCoV in cells and MHV-A59 in mice) reproducibility under regular infection with the same virus inoculum, (iii) DVGs can be synthesized from the full-length coronavirus genome, (iv) the sequences flanking the recombination point of DVGs are AU-rich and thus may contribute to the recombination events during gene expression, (v) the species and amounts of DVG are altered under different infection environments, and (vi) the biological nature of DVGs between in vitro and in vivo is similar. CONCLUSIONS: The identified biological characteristics of coronavirus DVGs in terms of abundance, reproducibility, and variety extend the current model for coronavirus gene expression. In addition, the biological features of alterations in amounts and species of coronavirus DVGs under different infection environments may assist the coronavirus to adapt to the altered environments for virus fitness and may contribute to the coronavirus pathogenesis. Consequently, the unveiled biological features may assist the community to study the gene expression mechanisms of DVGs and their roles in pathogenesis, contributing to the development of antiviral strategy and public health.

Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry.

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA-protein pull-down assay, six DVG-encoded proteins were identified by LC-MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.

Targeting the conserved coronavirus octamer motif GGAAGAGC is a strategy for the development of coronavirus vaccine.

BACKGROUND: Coronaviruses are pathogens of humans and animals that cause widespread and costly diseases. The development of effective strategies to combat the threat of coronaviruses is therefore a top priority. The conserved coronavirus octamer motif 5'GGAAGAGC3' exists in the 3' untranslated region of all identified coronaviruses. In the current study, we aimed to examine whether targeting the coronavirus octamer motif GGAAGAGC is a promising approach to develop coronavirus vaccine. METHODS: Plaque assays were used to determine the titers of mouse hepatitis virus (MHV)-A59 octamer mutant (MHVoctm) and wild-type (wt) MHV-A59 (MHVwt). Western blotting was used for the determination of translation efficiency of MHVoctm and MHVwt. Plaque assays and RT-qPCR were employed to examine whether MHVoctm was more sensitive to interferon treatment than MHVwt. Weight loss, clinical signs, survival rate, viral RNA detection and histopathological examination were used to evaluate whether MHVoctm was a vaccine candidate against MHVwt infection in BALB/c mice. RESULTS: In this study, we showed that (i) the MHVoctm with mutation of coronavirus octamer was able to grow to high titers but attenuated in mice, (ii) with the reduced multiplicity of infection (MOI), the difference in gene expression between MHVoctm and MHVwt became more evident in cultured cells, (iii) MHVoctm was more sensitive to interferon treatment than MHVwt and (iv) mice inoculated with MHVoctm were protected from MHVwt infection. CONCLUSIONS: Based on the results obtained from cultured cells, it was suggested that the synergistic effects of octamer mutation, multiplicity of infection and immune response may be a mechanism explaining the distinct phenotypes of octamer-mutated coronavirus in cell culture and mice. In addition, targeting the conserved coronavirus octamer motif is a strategy for development of coronavirus vaccine. Since the conserved octamer exists in all coronaviruses, this strategy of targeting the conserved octamer motif can also be applied to other human and animal coronaviruses for the development of coronavirus vaccines, especially the emergence of novel coronaviruses such as SARS-CoV-2, saving time and cost for vaccine development and disease control.

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo.

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains. RESULTS: In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions. CONCLUSIONS: The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.

Identification of subgenomic mRNAs derived from the coronavirus 1a/1b protein gene: Implications for coronavirus transcription.

Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.