Variations in the persistence of 5'-end genomic and subgenomic SARS-CoV-2 RNAs in wastewater from aircraft, airports and wastewater treatment plants.

Wastewater sequencing has become a powerful supplement to clinical testing in monitoring SARS-CoV-2 infections in the post-COVID-19 pandemic era. While its applications in measuring the viral burden and main circulating lineages in the community have proved their efficacy, the variations in sequencing quality and coverage across the different regions of the SARS-CoV-2 genome are not well understood. Furthermore, it is unclear how different sample origins, viral extraction and concentration methods and environmental factors impact the reads sequenced from wastewater. Using high-coverage, amplicon-based, paired-end read sequencing of viral RNA extracted from wastewater collected directly from aircraft, pooled from different aircraft and airport buildings or from regular wastewater plants, we assessed the genome coverage across the sample groups with a focus on the 5'-end region covering the leader sequence and investigated whether it was possible to detect subgenomic RNA from viral material recovered from wastewater. We identified distinct patterns in the persistence of the different genomic regions across the different types of wastewaters and the existence of chimeric reads mapping to non-amplified regions. Our findings suggest that preservation of the 5'-end of the genome and the ability to detect subgenomic RNA reads, though highly susceptible to environment and sample processing conditions, may be indicative of the quality and amount of the viral RNA present in wastewater.

Utility of SARS-CoV-2 Subgenomic RNA in Kidney Transplant Recipients Receiving Remdesivir.

INTRODUCTION: There is no reliable microbiological marker to guide responses to antiviral treatment in kidney transplant recipients (KTR) with COVID-19. We aimed to evaluate the dynamics of subgenomic RNA (sgRNA) RT-PCR before and after receiving treatment with remdesivir compared with genomic RNA (gRNA) RT-PCR and its use as a surrogate marker of viral replication. METHODS: We analyzed gRNA and sgRNA at baseline and after remdesivir treatment in KTR who received remdesivir for SARS-CoV-2 infection from November 2021 to February 2022. RESULTS: Thirty-four KTR received remdesivir for SARS-CoV-2 infection. The median time since transplantation was 80 months (IQR 3-321) and 75% of patients had previously received 3 doses of a mRNA SARS-CoV-2 vaccine. Three patients (8%) were classified with mild, 25 (73%) with moderate, and 6 (17%) with severe SARS-CoV-2 infection. Thirty-two (94%) patients received 5 doses of remdesivir and two patients received 2 doses. The median time between symptom onset to remdesivir treatment was 5 days (IQR 3-8.5). The median days of hospitalization were 6 (IQR 2-112). gRNA was positive in all patients at baseline and after remdesivir. Five (15%) patients had negative sgRNA at baseline and 20 (59%) after receiving remdesivir. Patients presenting with negative sgRNA at baseline were discharged from hospital in ≤ 6 days without complications. Moreover, those with negative sgRNA after remdesivir therapy did not require ICU admission and had favorable outcomes. Nevertheless, patients with positive sgRNA after antiviral treatment presented worse outcomes, with 47% requiring ICU admission and the three (9%) recorded deaths in the study were in this group. CONCLUSIONS: Based on these data, we hypothesize that sgRNA may have clinical utility to help monitor virologic response more accurately than gRNA in KTR who receive remdesivir. Moreover, patients with negative sgRNA at baseline may not require antiviral treatment and others presenting positive sgRNA at day 5 could benefit from prolonged or combined therapies.

Quantitative and qualitative subgenomic RNA profiles of SARS-CoV-2 in respiratory samples: A comparison between Omicron BA.2 and non-VOC-D614G.

The SARS-CoV-2 Omicron variants are notorious for their transmissibility, but little is known about their subgenomic RNA (sgRNA) expression. This study applied RNA-seq to delineate the quantitative and qualitative profiles of canonical sgRNA of 118 respiratory samples collected from patients infected with Omicron BA.2 and compared with 338 patients infected with non-variant of concern (non-VOC)-D614G. A unique characteristic profile depicted by the relative abundance of 9 canonical sgRNAs was reproduced by both BA.2 and non-VOC-D614G regardless of host gender, age and presence of pneumonia. Remarkably, such profile was lost in samples with low viral load, suggesting a potential application of sgRNA pattern to indicate viral activity of individual patient at a specific time point. A characteristic qualitative profile of canonical sgRNAs was also reproduced by both BA.2 and non-VOC-D614G. The presence of a full set of canonical sgRNAs carried a coherent correlation with crude viral load (AUC â€‹= â€‹0.91, 95% CI 0.88-0.94), and sgRNA ORF7b was identified to be the best surrogate marker allowing feasible routine application in characterizing the infection status of individual patient. Further potentials in using sgRNA as a target for vaccine and antiviral development are worth pursuing.

Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Expression dynamics of the aplysia abyssovirus.

Two recent studies documented the genome of a novel, extremely large (35.9 kb), nidovirus in RNA sequence databases from the marine neural model Aplysia californica. The goal of the present study was to document the distribution and transcriptional dynamics of this virus, Aplysia abyssovirus 1 (AAbV), in maricultured and wild animals. We confirmed previous findings that AAbV RNA is widespread and reaches extraordinary levels in apparently healthy animals. Transmission electron microscopy identified viral replication factories in ciliated gill epithelial cells but not in neurons where viral RNA is most highly expressed. Viral transcripts do not exhibit evidence of discontinuous RNA synthesis as in coronaviruses but are consistent with production of a single leaderless subgenomic RNA, as in the Gill-associated virus of Penaeus monodon. Splicing patterns in chronically infected adults suggested high levels of defective genomes, possibly explaining the lack of obvious disease signs in high viral load animals.

SARS-CoV-2 subgenomic RNA: formation process and rapid molecular diagnostic methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which caused coronavirus disease-2019 (COVID-19) is spreading worldwide and posing enormous losses to human health and socio-economic. Due to the limitations of medical and health conditions, it is still a huge challenge to develop appropriate discharge standards for patients with COVID-19 and to use medical resources in a timely and effective manner. Similar to other coronaviruses, SARS-CoV-2 has a very complex discontinuous transcription process to generate subgenomic RNA (sgRNA). Some studies support that sgRNA of SARS-CoV-2 can only exist when the virus is active and is an indicator of virus replication. The results of sgRNA detection in patients can be used to evaluate the condition of hospitalized patients, which is expected to save medical resources, especially personal protective equipment. There have been numerous investigations using different methods, especially molecular methods to detect sgRNA. Here, we introduce the process of SARS-CoV-2 sgRNA formation and the commonly used molecular diagnostic methods to bring a new idea for clinical detection in the future.

Differential Transcriptomic Landscapes of SARS-CoV-2 Variants in Multiple Organs from Infected Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the persistent coronavirus disease 2019 (COVID-19) pandemic, which has resulted in millions of deaths worldwide and brought an enormous public health and global economic burden. The recurring global wave of infections has been exacerbated by growing variants of SARS-CoV-2. In this study, the virological characteristics of the original SARS-CoV-2 strain and its variants of concern (VOCs; including Alpha, Beta, and Delta) in vitro, as well as differential transcriptomic landscapes in multiple organs (lung, right ventricle, blood, cerebral cortex, and cerebellum) from the infected rhesus macaques, were elucidated. The original strain of SARS-CoV-2 caused a stronger innate immune response in host cells, and its VOCs markedly increased the levels of subgenomic RNAs, such as N, Orf9b, Orf6, and Orf7ab, which are known as the innate immune antagonists and the inhibitors of antiviral factors. Intriguingly, the original SARS-CoV-2 strain and Alpha variant induced larger alteration of RNA abundance in tissues of rhesus monkeys than Beta and Delta variants did. Moreover, a hyperinflammatory state and active immune response were shown in the right ventricles of rhesus monkeys by the up-regulation of inflammation- and immune-related RNAs. Furthermore, peripheral blood may mediate signaling transmission among tissues to coordinate the molecular changes in the infected individuals. Collectively, these data provide insights into the pathogenesis of COVID-19 at the early stage of infection by the original SARS-CoV-2 strain and its VOCs.

A culture-free method for rapidly and accurately quantifying active SARS-CoV-2.

Determining the quantity of active virus is the most important basis to judge the risk of virus infection, which usually relies on the virus median tissue culture infectious dose (TCID50) assay performed in a biosafety level 3 laboratory within 5-7 days. We have developed a culture-free method for rapid and accurate quantification of active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by targeting subgenomic RNA (sgRNA) based on reverse transcription digital PCR (RT-dPCR). The dynamic range of quantitative assays for sgRNA-N and sgRNA-E by RT-dPCR was investigated, and the result showed that the limits of detection (LoD) and quantification (LoQ) were 2 copies/reaction and 10 copies/reaction, respectively. The delta strain (NMDC60042793) of SARS-CoV-2 was cultured at an average titer of 106.13 TCID50/mL and used to evaluate the developed quantification method. Copy number concentrations of the cultured SARS-CoV-2 sgRNA and genomic RNA (gRNA) gave excellent linearity (R2 = 0.9999) with SARS-CoV-2 titers in the range from 500 to 105 TCID50/mL. Validation of 63 positive clinical samples further proves that the quantification of sgRNA-N by RT-dPCR is more sensitive for active virus quantitative detection. It is notable that we can infer the active virus titer through quantification of SARS-CoV-2 sgRNA based on the linear relationship in a biosafety level 2 laboratory within 3 h. It can be used to timely and effectively identify infectious patients and reduce unnecessary isolation especially when a large number of COVID-19 infected people impose a burden on medical resources.

Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

Comparative subgenomic mRNA profiles of SARS-CoV-2 Alpha, Delta and Omicron BA.1, BA.2 and BA.5 sub-lineages using Danish COVID-19 genomic surveillance data.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread worldwide in the population since it was first detected in late 2019. The transcription and replication of coronaviruses, although not fully understood, is characterised by the production of genomic length RNA and shorter subgenomic RNAs to make viral proteins and ultimately progeny virions. Observed levels of subgenomic RNAs differ between sub-lineages and open reading frames but their biological significance is presently unclear. METHODS: Using a large and diverse panel of virus sequencing data produced as part of the Danish COVID-19 routine surveillance together with information in electronic health registries, we assessed the association of subgenomic RNA levels with demographic and clinical variables of the infected individuals. FINDINGS: Our findings suggest no significant statistical relationship between levels of subgenomic RNAs and host-related factors. INTERPRETATION: Differences between lineages and subgenomic ORFs may be related to differences in target cell tropism, early virus replication/transcription kinetics or sequence features. FUNDING: The author(s) received no specific funding for this work.

Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

Total and Subgenomic RNA Viral Load in Patients Infected With SARS-CoV-2 Alpha, Delta, and Omicron Variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic and subgenomic RNA levels are frequently used as a correlate of infectiousness. The impact of host factors and SARS-CoV-2 lineage on RNA viral load is unclear. METHODS: Total nucleocapsid (N) and subgenomic N (sgN) RNA levels were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in specimens from 3204 individuals hospitalized with coronavirus disease 2019 (COVID-19) at 21 hospitals. RT-qPCR cycle threshold (Ct) values were used to estimate RNA viral load. The impact of time of sampling, SARS-CoV-2 variant, age, comorbidities, vaccination, and immune status on N and sgN Ct values were evaluated using multiple linear regression. RESULTS: Mean Ct values at presentation for N were 24.14 (SD 4.53) for non-variants of concern, 25.15 (SD 4.33) for Alpha, 25.31 (SD 4.50) for Delta, and 26.26 (SD 4.42) for Omicron. N and sgN RNA levels varied with time since symptom onset and infecting variant but not with age, comorbidity, immune status, or vaccination. When normalized to total N RNA, sgN levels were similar across all variants. CONCLUSIONS: RNA viral loads were similar among hospitalized adults, irrespective of infecting variant and known risk factors for severe COVID-19. Total N and subgenomic RNA N viral loads were highly correlated, suggesting that subgenomic RNA measurements add little information for the purposes of estimating infectivity.

Identification of consensus hairpin loop structure among the negative sense subgenomic RNAs of SARS-CoV-2.

Background: SARS-CoV-2 is the causative agent of worldwide pandemic disease coronavirus disease 19. SARS-CoV-2 bears positive sense RNA genome that has organized and complex pattern of replication/transcription process including the generation of subgenomic RNAs. Transcription regulatory sequences have important role in the pausing of replication/transcription and generation of subgenomic RNAs. Results: In the present bioinformatics analysis, a consensus secondary structure was identified among negative sense subgenomic RNAs of SARS-CoV-2. This consensus region is present at the adjacent of initiation codon. Conclusions: This study proposed that consensus structured domain could involve in mediating the long pausing of replication/transcription complex and responsible for subgenomic RNA production. Supplementary Information: The online version contains supplementary material available at 10.1186/s42269-023-01002-3.

Successful lung transplantation using an allograft from a COVID-19-recovered donor: a potential role for subgenomic RNA to guide organ utilization.

Although the risk of SARS-CoV-2 transmission through lung transplantation from acutely infected donors is high, the risks of virus transmission and long-term lung allograft outcomes are not as well described when using pulmonary organs from COVID-19-recovered donors. We describe successful lung transplantation for a COVID-19-related lung injury using lungs from a COVID-19-recovered donor who was retrospectively found to have detectable genomic SARS-CoV-2 RNA in the lung tissue by multiple highly sensitive assays. However, SARS-CoV-2 subgenomic RNA (sgRNA), a marker of viral replication, was not detectable in the donor respiratory tissues. One year after lung transplantation, the recipient has a good functional status, walking 1 mile several times per week without the need for supplemental oxygen and without any evidence of donor-derived SARS-CoV-2 transmission. Our findings highlight the limitations of current clinical laboratory diagnostic assays in detecting the persistence of SARS-CoV-2 RNA in the lung tissue. The persistence of SARS-CoV-2 RNA in the donor tissue did not appear to represent active viral replication via sgRNA testing and, most importantly, did not negatively impact the allograft outcome in the first year after lung transplantation. sgRNA is easily performed and may be a useful assay for assessing viral infectivity in organs from donors with a recent infection.

Qualitative Subgenomic RNA to Monitor the Response to Remdesivir in Hospitalized Patients With Coronavirus Disease 2019: Impact on the Length of Hospital Stay and Mortality.

BACKGROUND: There is no reliable microbiological marker to guide the indication and the response to antiviral treatment in patients with coronavirus disease 2019 (COVID-19). We aimed to evaluate the dynamics of subgenomic RNA (sgRNA) in patients with COVID-19 before and after receiving treatment with remdesivir. METHODS: We included consecutive patients admitted for COVID-19 who received remdesivir according to our institutional protocol and accepted to participate in the study. A nasopharyngeal swab for quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was collected at baseline and after 3 and 5 days of treatment with remdesivir. Genomic and sgRNA were analyzed in those samples and main comorbidities and evolution were collected for the analyses. The main outcomes were early discharge (≤10 days) and 30-day mortality. RESULTS: A total of 117 patients were included in the study, of whom 24 had a negative sgRNA at baseline, with 62.5% (15/24) receiving early discharge (≤10 days) and no deaths in this group. From the 93 remaining patients, 62 had a negative sgRNA at day 5 with 37/62 (59.6%) with early discharge and a mortality rate of 4.8% (3/62). In the subgroup of 31 patients with positive sgRNA after 5 days of remdesivir, the early discharge rate was 29% (9/31) and the mortality rate was 16.1% (5/31). In multivariable analyses, the variables associated with early discharge were negative sgRNA at day 3 and not needing treatment with corticosteroids or intensive care unit admission. CONCLUSIONS: Qualitative sgRNA could help in monitoring the virological response in patients who receive remdesivir. Further studies are needed to confirm these findings.

Characterizing SARS-CoV-2 Transcription of Subgenomic and Genomic RNAs During Early Human Infection Using Multiplexed Droplet Digital Polymerase Chain Reaction.

BACKGROUND: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission requires understanding SARS-CoV-2 replication dynamics. METHODS: We developed a multiplexed droplet digital polymerase chain reaction (ddPCR) assay to quantify SARS-CoV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from genomic RNAs (gRNAs). We applied the assay to specimens from 144 people with single nasopharyngeal samples and 27 people with >1 sample. Results were compared to quantitative PCR (qPCR) and viral culture. RESULTS: sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio sgRNA:gRNA was stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Daily testing of 6 persons revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. sgRNA:gRNA is constant during infection despite changes in viral culture. CONCLUSIONS: Ct values from qPCR correlate with active viral replication. More work is needed to understand why some cultures are negative despite presence of sgRNA.

Subgenomic RNA Abundance Relative to Total Viral RNA Among Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate actively replicating virus, but sgRNA abundance has not been systematically compared between SARS-CoV-2 variants. sgRNA was quantified in 169 clinical samples by real-time reverse-transcription polymerase chain reaction, demonstrating similar relative abundance among known variants. Thus, sgRNA detection can identify individuals with active viral replication regardless of variant.

Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir.

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.

Cell Entry and Unusual Replication of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 is the causative virus for the CoVID-19 pandemic that has frequently mutated to continue to infect and resist available vaccines. Emerging new variants of the virus have complicated notions of immunity conferred by vaccines versus immunity that results from infection. While we continue to progress from epidemic to endemic as a result of this collective immunity, the pandemic remains a morbid and mortal problem. OBJECTIVE: The SARS-CoV-2 virus has a very complex manner of replication. The spike protein, one of the four structural proteins of the encapsulated virus, is central to the ability of the virus to penetrate cells to replicate. The objective of this review is to summarize these complex features of viral replication. METHODS: A review of the recent literature was performed on the biology of SARS-CoV-2 infection from published work from PubMed and works reported to preprint servers, e.g., bioRxiv and medRxiv. RESULTS AND CONCLUSION: The complex molecular and cellular biology involved in SARS-CoV-2 replication and the origination of >30 proteins from a single open reading frame (ORF) have been summarized, as well as the structural biology of spike protein, a critical factor in the cellular entry of the virus, which is a necessary feature for it to replicate and cause disease.

Translation landscape of SARS-CoV-2 noncanonical subgenomic RNAs.

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a positive-stranded RNA genome. Current proteomic studies of SARS-CoV-2 mainly focus on the proteins encoded by its genomic RNA (gRNA) or canonical subgenomic RNAs (sgRNAs). Here, we systematically investigated the translation landscape of SARS-CoV-2, especially its noncanonical sgRNAs. We first constructed a strict pipeline, named vipep, for identifying reliable peptides derived from RNA viruses using RNA-seq and mass spectrometry data. We applied vipep to analyze 24 sets of mass spectrometry data related to SARS-CoV-2 infection. In addition to known canonical proteins, we identified many noncanonical sgRNA-derived peptides, which stably increase after viral infection. Furthermore, we explored the potential functions of those proteins encoded by noncanonical sgRNAs and found that they can bind to viral RNAs and may have immunogenic activity. The generalized vipep pipeline is applicable to any RNA viruses and these results have expanded the SARS-CoV-2 translation map, providing new insights for understanding the functions of SARS-CoV-2 sgRNAs.