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Analyze the Data: Using RNAi RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by short double-stranded RNA molecules called siRNA (small interfering RNAs). In mammalian cells, transfection of 21–22 nucleotide siRNAs leads to degradation of mRNA molecules that contain the same sequence as the siRNA. In the following experiment, siRNA and knockout mice are used to investigate two related cell surface proteins designated p24 and p25 that are suspected to be cellular receptors for the uptake of a newly isolated virus. a. To test the efficacy of RNAi in cells, siRNAs specific to cell surface proteins p24 (siRNA–p24) and p25 (siRNA–p25) are transfected individually into cultured mouse cells. RNA is extracted from these transfected cells and the mRNA for proteins p24 and p25 are detected on Northern blots using labeled p24 cDNA or p25 cDNA as probes. The control for this experiment is a mock transfection with no siRNA. What do you conclude from this Northern blot about the specificity of the siRNAs for their target mRNAs? b. Next, the ability of siRNAs to inhibit viral replication is investigated. Cells are transfected with siRNA–p24 or siRNA–p25 or with siRNA to an essential viral protein. Twenty hours later, transfected cells are infected with the virus. After a further incubation period, the cells are collected and lysed. The number of viruses produced by each culture is shown below. The control is a mock transfection with no siRNA. What do you conclude about the role of p24 and p25 in the uptake of the virus? Why might the siRNA to the viral protein be more effective than siRNA to the receptors in reducing the number of viruses? Cell Treatment Control siRNA–p24 siRNA–p25 siRNA–p24 and siRNA–p25 siRNA to viral protein Number of Viruses/ml 1 107 3 106 2 106 1 104 1 102 c. To investigate the role of proteins p24 and p25 for viral replication in live mice, transgenic mice that lack genes for p24 or p25 are generated. The loxP-Cre conditional knock- out system is used to selectively delete the genes in cells of either the liver or the lung. Wild type and knockout mice are infected with virus. After a 24-hour incubation period, mice are killed and lung and liver tissues are removed and exam- ined for the presence (infected) or absence (normal) of virus by immunohistochemistry. What do these data indicate about the cellular requirements for viral infection in different tissues? Tissue Examined Mouse Liver Lung Wild type Knockout of p24 in liver Knockout of p24 in lung Knockout of p25 in liver Knockout of p25 in lung infected normal infected infected infected infected infected infected infected normal d. By performing Northern blots on different tissues from wild-type mice, you find that p24 is expressed in the liver but not in the lung, whereas p25 is expressed in the lung but not the liver. Based on all the data you have collected, propose a model to explain which protein(s) are involved in the virus entry into liver and lung cells. Would you predict that the cultured mouse cells used in parts (a) and (b) express p24, p25, or both proteins? Solution a. The Northern blot reveals the steady state mRNA levels for p24 (in the center) and p25 (on the right). For p24, only the transfection of siRNA-p24 reduces the amount of p24 mRNA; whereas the control transfection without siRNA and transfection with siRNA-p25 did not affect p24 mRNA levels. Similarly, for p25, only siRNAp25 reduced p25 RNA levels. These results demonstrate that the siRNAs can cause specific degradation of their target RNAs. b. In the cultured cells, transfection of either siRNA-p24 or siRNA-p25 yielded a viral titer that was slightly lower than the control transfection. This result indicates that reduction of either p24 mRNA or p25 mRNA (and presumably the proteins encoded by them) only minimally affects the ability of the virus to infect the cells. However, transfection of both siRNA-p24 and siRNA-p25 did result in a significant reduction in viral titer. In combination with the previous results, these results indicate that either p24 or p25 can be used as a viral receptor. You could hypothesize that loss of p24 and p25 mRNA would lead to a decrease in p24 and p25 protein, resulting in inhibition of viral infection and replication. Transfection of siRNA to a viral protein resulted in an even greater reduction of virus titer compared to transfection with siRNA to both p24 and p25. One possible explanation is that transfection with both either siRNA-p24 or siRNA-p25 leads to a reduction but not a complete elimination of cell surface receptors for the virus, whereas siRNA to a viral protein can inhibit viral replication/assembly directly. A second possibility has to do with the half-life of p24 and p25 proteins. A reduction of p24 and p25 mRNA may not lead to an immediate reduction in p24/p25 protein due to the stability of the protein. The protein level is determined by the half-life of the protein and the length of time after transfection of siRNA. For example, if the half lives of p24 and p25 proteins are greater than 24 hours, then during the 20 hour incubation after transfection with siRNA there should still be more than half of the protein still present. In this problem no information about the half-life of the protein was given, so a definitive conclusion cannot be made. But based on the results, it is reasonable to conclude that the half-life of the p24 and p25 protein is relatively long, such that 20 hours after transfection with the siRNA, sufficient protein remains to act as a receptor for the virus. c. Using the loxP-Cre system, the p24 or p25 gene can be selectively knocked-out in the liver or lung of transgenic mice. Using this approach, the receptor required for viral infection in specific tissues can be examined in tissue sections by immunohistochemistry. In the control, infection of a wild type mouse resulted in viral infection in both liver and lung tissue. In the transgenic mice lacking the p24 gene in lung, viral infection was seen in both the liver and lung. These results indicate that p24 protein is not required for viral infection of lung tissue. We cannot determine from this one piece of data alone whether or not p24 is required in the liver, as these mice still have a functional p24 gene in the liver. However, the mice which have p24 knocked out in the liver had no viral infection in the liver, indicating that p24 is required for viral infection in the liver. These mice show the expected viral infection of the lung, because their lung tissue was not affected by the selective knockout of p24 in the liver. Similarly, mice with a knockout of the p25 gene in liver displayed infection of the liver, demonstrating that p25 is not required for viral infection in the liver. Mice with a knockout of the p25 gene in lung had no viral infection in the lung, indicating that p25 is required for viral infection in lung. These results are somewhat unexpected because the cell culture experiment in part b indicated that either p24 or p25 could be used as a receptor for viral infection. But these knockout experiments indicate that in liver tissue, removal of p24 function alone is sufficient to prevent infection. Likewise, in lung tissue, removal of p25 function alone is sufficient to prevent infection. d. With the added information that liver tissue expresses only p24 and lung tissue expresses only p25, the apparent discrepancy between the results from the knockout mouse study and the cell culture studies can be resolved. Both the p24 and p25 protein can be used as a viral receptor in cells. But both proteins are not expressed in liver tissue; only p24 protein is present. Because p25 is absent, p24 is the protein used in liver as the viral receptor, and knockout of p24 in liver blocks viral infection. Similarly, in the lung, p25 is expressed, but p24 is not. Thus in lung, p25 is the protein used as the viral receptor, and knockout of p25 in lung blocks viral infection. The Northern blot in part a shows that in the cultured cells both p24 and p25 are present. Thus in the cultured cells both p24 and p25 can act as the viral receptor protein.