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

ABSTRACT: 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.

SUBMITTER: Lin CH

PROVIDER: S-EPMC10704767 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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

Lin Ching-Hung CH Hsieh Feng-Cheng FC Lai Chien-Chen CC Wang Wei-Chen WC Kuo Cheng-Yu CY Yang Chun-Chun CC Hsu Hsuan-Wei HW Tam Hon-Man-Herman HM Yang Cheng-Yao CY Wu Hung-Yi HY

Virology journal 20231207 1

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 p ...[more]

PMID: 38062493

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Project description:BackgroundThe mechanism by which coronavirus-defective viral genomes (DVGs) affect coronavirus and host cells during infection remains unclear. A variety of DVGs with different RNA structures can be synthesized from coronavirus-infected cells, and these DVGs can also encode proteins. Consequently, in the present study, we first dissected the effects of individual DVGs on the synthesis of IFNβ and ISG15 mRNAs at the RNA, protein and combined levels, and then examined whether different coronavirus-DVGs have different effects on the synthesis of IFNβ and ISG15 mRNAs and coronavirus replication both individually and collectively under different infection conditions.MethodsTo dissect the effects of individual DVGs on the synthesis of IFNβ and ISG15 mRNAs at the RNA, protein and combined levels, DVG 2.2 and DVG 5.1, which were previously identified in coronavirus-infected cells, and their mutants were constructed followed by transfection. Western blot and RT‒qPCR were used to detect the synthesis of protein and to quantify the synthesis of IFNβ and ISG15 mRNAs, respectively. To examined whether different coronavirus-DVGs have different effects on the synthesis of IFNβ and ISG15 mRNAs and coronavirus replication both individually and collectively under different infection conditions, different naturally occurring DVGs were selected and constructed followed by transfection after or before coronavirus infection and by RT‒qPCR and hemagglutination assay.ResultsThese results suggested that (i) coronavirus-DVGs at the RNA, protein and combined levels have different effects on the synthesis of IFNβ and ISG15 mRNAs, (ii) coronavirus-DVGs can inhibit coronavirus replication at least partly through interferon signaling and (iii) different DVGs have different effects on the synthesis of IFNβ and ISG15 mRNAs and coronavirus replication both individually and collectively under different infection conditions.ConclusionsCoronavirus replication can be regulated by diverse coronavirus-derived DVGs at least partly through innate immunity. Such regulation may contribute to the pathogenesis of coronavirus. The DVG populations in coronavirus-infected cells with the ability to inhibit coronavirus replication are expected to be potential resources for the identification of antivirals at the level of RNA, protein or in combination, and the methods used in the current study can be used as a platform for this purpose.

Project description:Ebola virus (EBOV), of the family Filoviridae, is an RNA virus that can cause a hemorrhagic fever with a high mortality rate. Defective viral genomes (DVGs) are truncated genomes that have been observed during multiple RNA virus infections, including in vitro EBOV infection, and have previously been associated with viral persistence and immunostimulatory activity. As DVGs have been detected in cells persistently infected with EBOV, we hypothesized that DVGs may also accumulate during viral replication in filovirus-infected hosts. Therefore, we interrogated sequence data from serum and tissue samples using a bioinformatics tool in order to identify the presence of DVGs in nonhuman primates (NHPs) infected with EBOV, Sudan virus (SUDV), or Marburg virus (MARV). Multiple 5' copy-back DVGs (cbDVGs) were detected in NHP serum during the acute phase of filovirus infection. While the relative abundance of total DVGs in most animals was low, serum collected during acute EBOV and SUDV infections, but not MARV infections, contained a higher proportion of short trailer sequence cbDVGs than the challenge stock. This indicated an accumulation of these DVGs throughout infection, potentially due to the preferential replication of short DVGs over the longer viral genome. Using reverse transcriptase PCR (RT-PCR) and deep sequencing, we also confirmed the presence of 5' cbDVGs in EBOV-infected NHP testes, which is of interest due to EBOV persistence in semen of male survivors of infection. This work suggests that DVGs play a role in EBOV infection in vivo and that further study will lead to a better understanding of EBOV pathogenesis. IMPORTANCE The study of filovirus pathogenesis is critical for understanding the consequences of infection and for the development of strategies to ameliorate future outbreaks. Defective viral genomes (DVGs) have been detected during EBOV infections in vitro; however, their presence in in vivo infections remains unknown. In this study, DVGs were detected in samples collected from EBOV- and SUDV-infected nonhuman primates (NHPs). The accumulation of these DVGs in the trailer region of the genome during infection indicates a potential role in EBOV and SUDV pathogenesis. In particular, the presence of DVGs in the testes of infected NHPs requires further investigation as it may be linked to the establishment of persistence.

Project description:BackgroundDefective 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.MethodsNanopore 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.ResultsThe 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.ConclusionsThe 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.

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

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

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