Requirement of the N-terminal region of nonstructural protein 1 in cis for SARS-CoV-2 defective RNA replication

Abstract

SARS-CoV-2 belongs to the family Coronaviridae and carries a single-stranded positive-sense RNA genome. During coronavirus (CoV) replication, defective or defective interfering RNAs that lack a large portion of the genome often emerge. These defective RNAs typically carry the necessary RNA elements that are required for replication and packaging. We identified the minimum requirement of the 5' proximal region necessary for viral RNA replication by using artificially generated SARS-CoV-2 minigenomes. The minigenomes consist of the 5'-proximal region, an open reading frame (ORF) that encodes a fusion protein consisting of the N-terminal of viral NSP1 and a reporter gene, and the 3' untranslated region of the SARS-CoV-2 genome. We used a modified SARS-CoV-2 variant to support replication of the minigenomes. A minigenome carrying the 5' proximal 634 nucleotides replicated, whereas those carrying shorter than 634 nucleotides did not, demonstrating that the entire 265 nt-long 5' untranslated region and N-terminal portion of the NSP1 coding region are required for the minigenome replication. Minigenome RNAs carrying a specific amino acid substitution or frame shift insertions in the partial NSP1 coding sequence abrogated minigenome replication. Introduction of synonymous mutations in the minigenome RNAs also affected the replication efficiency of the minigenomes. These data suggest that the expression of the N-terminal portion of NSP1 and the primary sequence of the 5' proximal 634 nucleotides are important for minigenome replication.IMPORTANCESARS-CoV-2, the causative agent of COVID-19, is highly transmissible and continues to have a significant impact on public health and the global economy. While several vaccines mitigate the severe consequences of SARS-CoV-2 infection, mutant viruses with reduced reactivity to current vaccines continue to emerge and circulate. This study aimed to identify the minimal 5' proximal region of SARS-CoV-2 genomic RNA required for SARS-CoV-2 defective RNA replication and investigate the importance of an ORF encoded in these defective RNAs. Identifying cis-acting replication signals of SARS-CoV-2 genomic RNA is critical for the development of antivirals that target these signals. Additionally, replication-competent defective RNAs can serve as therapeutic reagents to interfere with SARS-CoV-2 replication. Our findings provide valuable insights into the mechanisms of SARS-CoV-2 RNA replication and the development of reagents that suppress SARS-CoV-2 replication.

Keywords: SARS-CoV-2; cis-acting replication signal; coronavirus.

Fig 1

Replication of SARS-CoV-2 minigenome RNAs. (A) A schematic representation of the RNA secondary structure of the 5′ proximal region of the SARS-CoV-2 genome determined by Huston et al. (17). (B) Schematic representation and replication of SARS-rep-rLuc RNAs in the absence and presence of helper virus. Vero3a/E cells were infected with SARS2-del3a/E virus or left uninfected and transfected with the indicated RNAs. The cells were harvested at 30 h p.i. and subjected to RNA extraction. Total RNA was analyzed by Northern blot using the rLuc probe and separated on an agarose gel for the detection of rRNAs. The integrity of in vitro synthesized RNA (input RNA) was validated by gel electrophoresis (bottom panels).

Fig 2

Identification of the minimum 5′ proximal region required for replication of the SARS-CoV-2 minigenome RNA. (A) Replication of SARS2-rep-rLuc variants. The sequence of the partial NSP1 encoded by the SARS2-rep-rLuc RNA variants is shown in the upper part. The 632-rLuc RNA carries two extra nucleotides indicated by small bold letters to encode the NSP1-rLuc fusion protein. Vero3a/E cells were infected with SARS2-del3a/E virus or left uninfected and transfected with the indicated RNAs. The cells were harvested at 30 h p.i. and subjected to RNA extraction. Total RNA was analyzed by Northern blot using the rLuc probe and separated on an agarose gel for the detection of rRNAs (middle panels). The integrity of in vitro synthesized RNA (input RNA) was validated by gel electrophoresis (bottom panel). (B) Expression of rLuc in cells transfected with the SARS2-rep-rLuc RNAs. VeroE6 cells were transfected with the indicated RNAs in the absence of helper virus infection. Cell lysates were harvested at 8 h post transfection and analyzed by rLuc assay (top panel) and Western blot using anti-rLuc and anti-tubulin antibodies (bottom panels). (C) Expression of rLuc in cells transfected with the SARS2-rep-rLuc RNAs in the absence or presence of helper virus. Vero-ORF3-E cells cells were infected with SARS-CoV-2-delORF3-E virus or left uninfected and transfected with the indicated RNAs. Cell lysates were harvested at 30 h p.i. and subjected to rLuc assay (left panel) and Western blot using anti-rLuc and anti-tubulin antibodies (right panel). *10-fold diluted sample.

Fig 3

Schematic representation of RNA secondary structures of SL1–SL5 of the SARS-CoV-2 genome and minigenome RNAs. (A) RNA secondary structures of the 5′ proximal region of the SARS-CoV-2 genome determined by Huston et al. (17). Predicted RNA secondary structures of the 5′ proximal region of 652-rLuc RNA (B), 652-rLuc-delSL4 (C), and 652-rLuc-delSL5 (D).

Fig 4

Schematic representation of RNA secondary structures of minigenome RNAs. (A) Predicted RNA secondary structure of the 5′ proximal region of 652-rLuc RNA. (B) Predicted RNA secondary structures of the 5′ proximal region of the 652-rLuc-opt RNA.

Fig 5

Replication of SARS2-rep-640-rLuc RNA variants. (A) Sequence of the SARS2-rep-640-rLuc RNA variants. Mutated sites are indicated by red squares. (B) Replication of the SARS-rep-640-rLuc RNA variants in the absence and presence of helper virus. Vero3a/E cells were infected with SARS2-del3a/E virus and transfected with the indicated RNAs. The cells were harvested at 30 h p.i. and subjected to RNA extraction. Total RNA was analyzed by Northern blot using the rLuc probe (top panel) and separated on an agarose gel for the detection of rRNAs (middle panel). The integrity of in vitro synthesized RNA (input RNA) was validated by gel electrophoresis (bottom panel). (C) Expression of rLuc in cells transfected with the SARS2-rep-640-rLuc RNA variants. VeroE6 cells were transfected with the indicated RNAs in the absence of the helper virus. Cell lysates were harvested at 8 h post transfection and analyzed by rLuc assay and Western blot using anti-rLuc and anti-tubulin antibodies.

Fig 6

Replication of SARS2-rep-652 RNA-based FS mutants. (A) Schematic representation and N-terminal amino acid sequence of the FS mutants. (B) Replication of the FS mutants in the absence and presence of the helper virus. Vero3a/E cells were infected with SARS2-del3a/E virus and transfected with the indicated RNAs. The cells were harvested at 30 h p.i. and subjected to RNA extraction. Total RNA was analyzed by Northern blot using the rLuc probe (left top panel) and separated on an agarose gel for the detection of rRNAs (left bottom panel). The integrity of in vitro synthesized RNA (input RNA) was validated by gel electrophoresis (right panel). (C) Expression of rLuc from the FS mutants. VeroE6 cells were transfected with the indicated RNAs. Cell lysates were harvested at 8 h post transfection and analyzed by rLuc assay (left panel) and Western blot using anti-rLuc and anti-tubulin antibodies (right panel).

Fig 7

Predicted RNA secondary structures of the 5′ proximal region of the SARS2-rep-652 RNA-based FS mutants.