key: cord-0781222-283llw7c authors: Nyayanit, Dimpal A.; Sarkale, Prasad; Shete-Aich, Anita; Kumar, Abhinendra; Patil, Savita; Majumdar, Triparna; Baradkar, Shreekant; Gawande, Pranita; Mohandas, Sreelekshmy; Yadav, Pragya D title: Possible role of accessory proteins in the viral replication for the 20I/501Y.V1 (B.1.1.7) SARS-CoV-2 variant date: 2021-05-03 journal: bioRxiv DOI: 10.1101/2021.04.30.442222 sha: c4ca82841b2b5088b68407120a60ccbfd1d808e1 doc_id: 781222 cord_uid: 283llw7c The study investigates the replication cycle and transcriptional pattern of the B.1.1.7 variant. It was observed that the B.1.1.7 variant required a longer maturation time. The transcriptional response demonstrated higher expression of ORF6 and ORF8 compared to nucleocapsid transcript till the eclipse period which might influence higher viral replication. The number of infectious viruses titer is higher in the B.1.1.7, despite a lesser copy number than B.1, indicating higher infectivity. The recent identification of the new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) variants has been of public health concern globally, after its first report from China in December 2019 (Zhu et al., 2020) . SARS-CoV-2 encodes for structural [E (envelope) , N (nucleocapsid), M (membrane), and S (spike)], a large polypeptide cleaved into different non-structural proteins and six accessory proteins (ORFs 3a, 6, 7a, 7b, 8, 10) (Zhou et al., 2020) . The expression of these proteins is responsible for the replication of the SARS-CoV-2 with its hosts. Information on the growth kinetics helps in understanding the virus-host interaction. It is also known that like other RNA viruses, The mutation rate of SARS-CoV-2 is higher in comparison to other virus groups (Duffy, 2018) . The growth kinetic study of the SARS-CoV demonstrated the viral release at 7th-hour post-infection (hpi) in the Vero-E6 cells (Keyaerts et al., 2005) . The growth kinetics study for the SARS-CoV-2 demonstrates a similar trend to SARS-CoV, where the virus was observed in the supernatant from 7 th hpi (Nyayanit et al., 2020) . Further, limited data is present on the transcriptional response of the SARS-CoV-2 proteins after it infects a host cell (Blanco-Melo et al., 2020; Krishnamoorthy et al., 2021; Nyayanit et al., 2020; Sun et al., 2020) at different time-points. Comparison of respiratory viruses with the SARS-CoV-2 in cell lines and animal systems revealed a reduced cytokine and Interferon (Type I and III) response (Blanco-Melo et al., 2020) . Transcriptome comparison of the SARS-CoV, MERS-CoV and the SARS-CoV-2 showed the suppression of the mitochondrial pathway and the glutathione metabolism (Krishnamoorthy et al., 2021) . Time-point analysis by Sun et al demonstrated early host response to the SARS-CoV-2 entry to the host cell in comparison to SARS-CoV and MERS-CoV (Sun et al., 2020) . Delayed appearance of the cytopathic effect for the B.1.1.7 variant, with similar passage history, in comparison to the B.1 variant prompted us to investigate further. This study was carried out to understand the time required to complete the replication cycle of the B.1.1.7, a 4 variant of concern (VOC). Further, in vitro transcriptional response generated at various time points was assessed. The intracellular live virus in pellet and extracellular live virus in supernatant was calculated in a triplicate set using endpoint titration assay in Vero CCL81 cells at defined time intervals. The presence of live virus was detected in pellet beginning from the 8 th hour's post-infection (hpi) while the virus was released in the supernatant only at 12 th hpi ( Figure 1) . The time lag of 3h till the first virus appearance in the supernatant was observed. In the case of the B.1 variant, the virus was observed in the supernatant approximately within an hour after its appearance in the cell pellet (Nyayanit et al., 2020) . The delayed observance of the B.1.1.7 in the supernatant in respect to B.1 led us to interpret that B.1.1.7 has a slow growth cycle caused due to its delayed maturation time. 5 In vitro transcriptional response for the SARS-CoV-2 genes was obtained using the nextgeneration sequencing of the cell pellet harvested at defined time intervals for the B.1.1.7 variant. The nucleocapsid transcript is expressed at a higher level in comparison to other viral transcripts at all-time points of experiments for SARS-CoV-2 (Finkel et al., 2020 , Nyayanit et al., 2020 . The RPKM values observed was hence normalized with the nucleocapsid expression. Figure 2A and B is the log10 plot of the normalized RPKM values obtained for structural protein transcript & non-structural polypeptide transcript and accessory protein transcript respectively at a defined time interval. The structural, nonstructural, and accessory protein transcripts of the B.1 virus were always expressed in lower levels as compared to nucleocapsid transcript (Nyayanit et al., 2020) . However, in the case of the B.1.1.7 variant, a few of the structural and accessory protein transcripts are expressed more than the nucleocapsid protein. The structural gene transcripts of B.1.1.7 are expressed in lower levels as compared to the nucleocapsid transcript at the time points (Figure 2A) . But, the accessory protein ORF8 and ORF10 transcripts are expressed in higher levels at every time point in the study. The ORF6, ORF7a and 7b transcript are expressed at higher levels till the eclipse period of the virus and drops later. The expression of the ORF3a transcript is at lower levels for each time point ( Figure 2B ). It has been reported earlier that pathogenic viruses increase viral replication by antagonizing the nuclear import to suppress the host immune response (Kato et al., 2021) . ORF6 protein of the SARS-CoV-2 bind to the Nup98-Rae1 thereby targeting nuclear import (Miorin et al., 2020) , indicated by its higher expression levels. Further, the role of ORF8 in viral replication was also reported (Muth et al., 2018) . This indicates that the higher level of ORF6 and ORF8 can be associated with higher viral replication. With a similar observation in the increased expression level of the ORF6 and ORF8 transcript, it could be hypothesized that B.1.1.7 virus has higher viral replication. Overall higher viral copy number and a lesser reduction in viral titer were observed for the B.1 variant. The viral titer of the B.1.1.7 variant drops at a higher rate in the consecutive days however the amount of the infectious virus is still larger in comparison to the B.1 variant indicating replication. Further the ratio of the non-infectious: infectious is approximately in 3:1 ratio this indicates the infectious virus available for infecting a host cell is more in the B.1.1.7 variant. This makes it highly transmissible leading to a higher number of COVID-19 cases (Davies et al., 2021) but having reduced pathogenicity to a lesser infectious viral ratio. This study reveals that B.1.1.7 SARS-CoV-2 variant is a slow-growing virus. Further, a higher replication along with reduced infectious viral titer was hypothesized to be linked to higher transmission with reduced pathogenicity. The present study had approval by the Institutional Animal Ethics and Biosafety Committee of Indian Council of Medical Research (ICMR) -National Institute of Virology (NIV), Pune. The 20I/501Y.V1 (B.1.1.7) SARS-CoV-2 isolate used in this study from the throat/nasal swab of United Kingdom traveller to India and submitted to GISAID (GISAID Number: EPI_ISL_825086) during late December 2020 (Yadav et al., 2021) . Vero CCL81 grown in the 24-well plate was infected with the 20I/501Y.V1 SARS-Cov-2 strain (TCID 50 :10 5.5 /ml, Vero CCL81 P-3). A 0.1 multiplicity of infection (MOI) was used to study the viral replication and the transcriptional response of the host Vero CCL81 cell line in a triplicate set. Cell pellet and supernatant were harvested at the defined time intervals (0, 30 min, 1h, 2h, 4h, 6h, 7h, 8h, 9h, 10h, 12h, 24h, 48 h, 72h, 96h, 120h and 144h) and stored at -80°C till further use. TCID 50 and Cyclic threshold (Ct) value for the E gene were determined calculated for both the supernatants and cell pellets using the end-point titration assay and real-time PCR method (Choudhary et al., 2020) . Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19 Development of in vitro transcribed RNA as positive control for laboratory diagnosis of SARS-CoV-2 in India Estimated transmissibility and impact of Why are RNA virus mutation rates so damn high? SARS-CoV-2 utilizes a multipronged strategy to suppress host protein synthesis Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex Growth kinetics of SARScoronavirus in Vero E6 cells Comparative transcriptome analysis of SARS-CoV, MERS-CoV, and SARS-CoV-2 to identify potential pathways for drug repurposing SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling Attenuation of replication by a 29 nucleotide deletion in SARScoronavirus acquired during the early stages of human-to-human transmission Transcriptome & viral growth analysis of SARS-CoV-2-infected Vero CCL-81 cells Transcriptome & viral growth analysis of SARS-CoV-2-infected Vero CCL-81 cells Comparative Transcriptome Analysis Reveals the Intensive Early Stage Responses of Host Cells to SARS-CoV-2 Infection MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferonα treatment Isolation and characterization of the new SARS-CoV-2 variant in travellers from the United Kingdom to India: VUI-202012/01 of the B.1.1.7 lineage Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin A Novel Coronavirus from Patients with Pneumonia in China No competing interest exists among the authors. The findings and conclusions are of the authors, and the funding agencies have no role in any part of the study. PDY contributed to study design, data analysis, interpretation and writing and critical review. DAN contributed to study design, data analysis and interpretation and writing. AMS, TP, SP, AK, PG and SM contributed to data collection and data interpretation. All the authors have read and approved the paper.