key: cord-0908165-yl3czlp8
authors: Verma, Rohit; Saha, Sandhini; Kumar, Shiv; Mani, Shailendra; Maiti, Tushar Kanti; Surjit, Milan
title: RNA-protein interaction analysis of SARS-CoV-2 5’- and 3’-untranslated regions identifies an antiviral role of lysosome-associated membrane protein-2
date: 2021-01-06
journal: bioRxiv
DOI: 10.1101/2021.01.05.425516
sha: 7eb9665f781eaad2a5e8c42c6a2acf50b40011bc
doc_id: 908165
cord_uid: yl3czlp8

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is a positive-strand RNA virus. Viral genome is capped at the 5’-end, followed by an untranslated region (UTR). There is poly-A tail at 3’-end, preceded by an UTR. Self-interaction between the RNA regulatory elements present within 5’- and 3’-UTRs as well as their interaction with host/virus-encoded proteins mediate the function of 5’- and 3’-UTRs. Using RNA-protein interaction detection (RaPID) assay coupled to liquid chromatography with tandem mass-spectrometry, we identified host interaction partners of SARS-CoV-2 5’- and 3’-UTRs and generated an RNA-protein interaction network. By combining these data with the previously known protein-protein interaction data proposed to be involved in virus replication, we generated the RNA-protein-protein interaction (RPPI) network, likely to be essential for controlling SARS-CoV-2 replication. Notably, bioinformatics analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism. Lysosome-associated membrane protein-2a (Lamp2a) was one of the host proteins that interact with the 5’-UTR. Further studies showed that Lamp2 level is upregulated in SARS-CoV-2 infected cells and overexpression of Lamp2a and Lamp2b variants reduced viral RNA level in infected cells and vice versa. In summary, our study provides an useful resource of SARS-CoV-2 5’- and 3’-UTR binding proteins and reveal the antiviral function of host Lamp2 protein. Importance Replication of a positive-strand RNA virus involves an RNA-protein complex consisting of viral genomic RNA, host RNA(s), virus-encoded proteins and host proteins. Dissecting out individual components of the replication complex will help decode the mechanism of viral replication. 5’- and 3’-UTRs in positive-strand RNA viruses play essential regulatory roles in virus replication. Here, we identified the host proteins that associate with the UTRs of SARS-CoV-2, combined those data with the previously known protein-protein interaction data (expected to be involved in virus replication) and generated the RNA-protein-protein interaction (RPPI) network. Analysis of the RPPI network revealed the enrichment of factors involved in translation initiation and RNA metabolism, which are important for virus replication. Analysis of one of the interaction partners of the 5’-UTR (Lamp2a) demonstrated its antiviral role in SARS-CoV-2 infected cells. Collectively, our study provides a resource of SARS-CoV-2 UTR-binding proteins and identifies an antiviral role of host Lamp2a protein.

 Table 1 

300+3'203 RPPI network is stronger than the 5'-300 network (Fig 2D, 2E ).

Recently protein interaction map of SARS-CoV-2 has been reported (25 Table 1 identified six proteins (RPL13, PDHA1, HABP4, MTHFD1, TUBA1A, 298 PLA2G4A), against whom known drugs exist (Table 2B) propionate. A recent report shows the ability of another steroid drug "Ciclesonide" to block 304 SARS-CoV-2 replication by targeting its replication-transcription complex (30). 

Further studies are required to understand the actual process. Nevertheless, the current 517 study identifies the repertoire of host proteins that associate with the SARS-CoV-2 5'-and

523 Sequence corresponding to 1-300 nucleotides at the 5' end (5'-300) and 29676-29900 at the considered for further studies (see Table 1 ). The mass spectrometry proteomics data have 

Evolution of the novel coronavirus 787 from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human 788 transmission

Syndrome Coronavirus 2: From Gene Structure to Pathogenic Mechanisms and 792 Potential Therapy

Characteristics of SARS-CoV-2 and COVID-19

Potential Causes and Consequences of Gastrointestinal 797 Disorders during a SARS-CoV-2 Infection

Novel human coronavirus (SARS-CoV-2): A lesson from 800 animal coronaviruses

An Updated Overview of Their 803

Coronaviruses 804 methods in Molecular Biology

Clustering analysis of correlation between biological replicates used in LC-MS-MS

Venny analysis of 5'-300 and 3'-203 RNA interacting proteins. (3'-203 in red, 3'-1016 203_Biotin in blue, 5'-300 in green, 5'-300_Botin in yellow

Schematic of RNA-protein interaction network of the 3'-203 SARS-CoV-2 RNA. Black 1019 node: 3'-203

Schematic of RNA-protein interaction network of the 5'-300 SARS-CoV-2 RNA. Black 1022 node: 5'-300; Blue node: Host protein

Schematic of RNA-protein interaction network of the 5

Black nodes: 5'-300 or 3'-203; Blue or red nodes: Host protein. Host proteins 1026 that interact with each other contain yellow or green color inside the node. Common 1027 host proteins that interact with both 5'-3000 and 3'-203 RNA are represented with SARS-CoV-2 for the indicated periods. Real-time PCR reactions were performed 1039 using SYBR green based protocol

Intracellular level of SARS-CoV-2 RNA in Vero E6 cells that are infected with SARS-S3 Fig. Normalized distribution curve of LC-MS-MS identified peptides

RPPI network of SARS-CoV-2 5'-300 RNA + 3'-203 RNA + PPI involved in virus 1183 replication

GO analysis of 5'-300 and 3'-203 SARS-CoV-2 RNA interacting proteins

Reactome pathway analysis of 5'-300 and 3'-203 SARS-CoV-2 RNA interacting 1186 proteins

Lamp2 modulates viral RNA level in SARS-CoV-2

List of host proteins that associate with SARS-CoV-2 5'-300 and 3'-203 RNA