questions Flashcards
(11 cards)
What is the primary function of the 5’-UTR in the SARS-CoV-2 RNA genome, and why is it a target for RBM24? (3 marks)
The 5’-UTR contains structured RNA elements, including stem-loops (SLs), which regulate the initiation of translation of viral polyproteins. (1 mark) RBM24 targets the 5’-UTR, specifically the GUGUG motif in SL4, to inhibit ribosome assembly and thereby block translation. (2 marks)
Describe how RBM24 affects SARS-CoV-2 replication at the level of protein synthesis. Include specific viral proteins discussed in the study. (4 marks)
RBM24 suppresses the translation of viral polyproteins by preventing the assembly of the 80S ribosome. (1 mark) This results in decreased levels of viral proteins such as Nsp3 and N protein, as demonstrated by Western blot analyses in H1299-ACE2 and A549-ACE2 cells. (3 marks)
Explain the significance of the GUGUG motif in the SL4 domain of the 5’-UTR with respect to RBM24 binding. (3 marks)
The GUGUG motif serves as a specific RNA sequence that RBM24 recognises and binds to. (1 mark) This interaction is crucial for RBM24’s ability to inhibit translation, as demonstrated by pulldown assays showing reduced binding to SL4 mutants lacking the GUGUG sequence. (2 marks)
Based on the results shown in Figure 1, how does RBM24 overexpression influence SARS-CoV-2 RNA and protein levels in H1299-ACE2 and A549-ACE2 cells? (4 marks)
Overexpression of RBM24 (pRBM24) significantly reduces SARS-CoV-2 RNA levels (as shown by RT-qPCR) (2 marks) and decreases the expression of viral proteins Nsp3 and N (as shown by Western blot analysis) in both cell lines, indicating that RBM24 inhibits viral replication and protein production. (2 marks)
In Figure 2, how do the deletion mutants of RBM24 (ΔRNP1, ΔRNP2, ΔRNP1/2) affect its ability to inhibit viral replication? What does this suggest about the functional domains of RBM24? (5 marks)
The deletion mutants ΔRNP1, ΔRNP2, and ΔRNP1/2 fail to reduce SARS-CoV-2 protein expression and RNA levels in comparison to the wild-type RBM24, as demonstrated by Western blot and RT-qPCR analyses. This indicates that both RNP1 and RNP2 domains are essential for RBM24’s antiviral activity, as removing either domain abolishes its ability to inhibit viral replication. This suggests that the RNP domains are crucial for binding to the 5’-UTR and blocking ribosome assembly, preventing translation of viral proteins.
Figure 1: How does RBM24 overexpression impact the production of infectious SARS-CoV-2 particles, and what evidence supports this conclusion?
RBM24 overexpression (pRBM24) significantly reduces the production of infectious SARS-CoV-2 particles, as shown by plaque assay in H1299-ACE2 cells (Figure 1f). The viral titre in the pRBM24 group was markedly lower compared to the pcDNA3.1 control, demonstrating that fewer infectious particles were released. This result is consistent with the observed decrease in viral protein levels (Nsp3, N) and viral RNA (Figure 1b and 1c), indicating that RBM24 inhibits viral replication and particle production by blocking translation.
Figure 1: Explain the experimental design used to assess the effects of RBM24 knockdown and overexpression on viral protein and RNA levels. How do the results validate RBM24’s role as a host restriction factor?
The experimental design involved transfecting H1299-ACE2 and A549-ACE2 cells with siRBM24 (knockdown) or pRBM24 (overexpression), followed by infection with SARS-CoV-2 (MOI 0.1). Protein and RNA analyses were conducted using Western blot and RT-qPCR, respectively. siRBM24 increased Nsp3, N protein, and viral RNA levels, whereas pRBM24 decreased them, validating RBM24’s role as a host restriction factor by negatively regulating viral replication.
How do the findings related to ΔRNP1, ΔRNP2, and ΔRNP1/2 mutants support the hypothesis that RNP domains are essential for RBM24’s antiviral activity? Provide specific data from both protein and RNA analyses.
The ΔRNP1, ΔRNP2, and ΔRNP1/2 mutants failed to reduce Nsp3 and N protein levels compared to wild-type RBM24 (Figure 2a). Additionally, RT-qPCR results (Figure 2b) show no significant reduction in viral RNA levels in mutant-transfected cells, indicating that both RNP1 and RNP2 domains are crucial for inhibiting viral replication. These findings underscore that intact RNP domains are necessary for RBM24 to bind the 5’-UTR and exert its antiviral function.
Figure 3: Describe how the RNA pulldown assay was utilised to confirm RBM24 binding specificity to the SARS-CoV-2 5’-UTR. What do the results indicate regarding the importance of the GUGUG motif?
The RNA pulldown assay involved incubating cell lysates with biotin-labelled RNA probes representing the SARS-CoV-2 5’-UTR, SL4, and mutated SL4 (GUGUG → GAGAG). Wild-type RBM24 bound strongly to the SL4 probe but not to the mutated version, demonstrating that the GUGUG motif is critical for binding. Additionally, yeast tRNA (negative control) showed no binding, confirming the sequence specificity of RBM24.
Figure 4: What evidence supports the conclusion that the GUGUG motif in SL4 is crucial for RBM24 binding? How do the authors demonstrate that this interaction is sequence-specific?
The pulldown assay in Figure 4e shows that RBM24 binds robustly to wild-type SL4 but not to the SL4 mutant (GUGUG → GAGAG), indicating that the GUGUG motif is necessary for interaction. Furthermore, the in vitro translation assay demonstrated reduced luciferase activity in SL4 mutants, linking GUGUG-specific binding to functional inhibition of translation.
Figure 4: The authors conducted a ribosome assembly assay to assess RBM24’s impact on ribosome formation. What were the findings, and how do they contribute to the proposed mechanism of translation inhibition?
