Long Noncoding RNA AROD Inhibits Host Antiviral Innate Immunity via the miR-324-5p–CUEDC2 Axis

ABSTRACT Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs that are involved in multiple biological processes. Here, we report a mechanism through which the lnc-AROD–miR-324-5p–CUEDC2 axis regulates the host innate immune response, using influenza A virus (IAV) as a model. We identified that host lnc-AROD without protein-coding capability is composed of 975 nucleotides. Moreover, lnc-AROD inhibited interferon-β expression, as well as interferon-stimulated genes ISG15 and MxA. Furthermore, in vivo assays confirmed that lnc-AROD overexpression increased flu virus pathogenicity and mortality in mice. Mechanistically, lnc-AROD interacted with miR-324-5p, leading to decreased binding of miR-324-5p to CUEDC2. Collectively, our findings demonstrated that lnc-AROD is a critical regulator of the host antiviral response via the miR-324-5p–CUEDC2 axis, and lnc-AROD functions as competing endogenous RNA. Our results also provided evidence that lnc-AROD serves as an inhibitor of the antiviral immune response and may represent a potential drug target. IMPORTANCE lnc-AROD is a potential diagnostic and discriminative biomarker for different cancers. However, so far the mechanisms of lnc-AROD regulating virus replication are not well understood. In this study, we identified that lnc-AROD is downregulated during RNA virus infection. We demonstrated that lnc-AROD enhanced CUEDC2 expression, which in turn inhibited innate immunity and favored IAV replication. Our studies indicated that lnc-AROD functions as a competing endogenous RNA that binds miR-324-5p and reduces its inhibitory effect on CUEDC2. Taken together, our findings reveal that lnc-AROD plays an important role during the host antiviral immune response.

The authors describe a mechanism through which the lnc22 AROD/miR-324-5p/CUEDC2 axis regulates the host innate immune response using 23 influenza A virus as a model. From this article the conclusion is that lnc-AROD is a critical regulator of the host antiviral response via the miR-324-5p/CUEDC2 axis and lnc-AROD functions as competing endogenous RNA.
I due have some questions/suggestions to clarify the methods and about the interpretation of the results: LINE 47= Is there a reference. If so, please add reference in this point.
LINE 275= Further example should be added, together with other references.
Reviewer #2 (Comments for the Author): In this study, the authors demonstrated an association between the abundance of lnc-AROD and the influenza A virus infection in both human cell lines and in the mouse model. Within the manuscript, the authors claimed the following findings: 1. Lnc-AROD interacts with miR-324-5p, and the interaction is potentially direct; 2. The above interaction leads to a decreased binding of miR-324-5p to the 3' UTR of CUEDC2 mRNA, which then causes an increased abundance of CUEDC2 protein; 3. Lnc-AROD is a critical regulator of the host antiviral response (Lnc-AROD abundance is positively associated with the virus titer post-IAV infection); 4. The antiviral function of lnc-AROD is realized through its direct interaction with miR-324-5p; 5. Through its regulatory effect on CUEDC2 via miR-324-5p, lnc-AROD abundance is negatively associated with the expression level of IFN-β, ISG15, and MxA. The authors showed experimental evidence to support the above findings from the perspectives of both ex vivo and in vivo. To test the suggested molecular mechanism of action in regulating the innate immune response to IAV by lnc-AROD, the authors conducted both overexpression and knockdown studies using multiple experimental methodologies. They provided solid results to support the conclusion of this manuscript, as well as the suggested working model described in Fig. 9. In Fig. 3&4, the authors showed that lnc-AROD abundance is associated with the infection rate of IAV, using both KD and overexpression of lnc-AROD. The experimental evidence was collected from both cultured cell lines and mouse models. Fig.5 showed that lnc-AROD has a suppressive effect on IFN-β, ISG15, and MxA, but not on IRF3 or TBK1. The authors should explain more on the rationale of the selection of genes being examined here in their study. Through Fig. 6&7, the authors showed evidence to support the hypothesis that lnc-AROD can form a direct interaction with miR-324-5p, and this interaction "competes" the affinity between miR and CUEDC2 mRNA. The authors then suggested that this competition may lead to a modified/regulated expression of downstream genes that play important roles in innate immunity. Moving on to Fig.8, the authors showed evidence that CUEDC2 itself has an effect on the expression level of the abovementioned three genes that can be impacted by the level of lnc-AROD. Moreover, the authors also presented data through the figures to show that viral titer can be impacted by the experimental treatment (such as overexpression of CUEDC2, etc), which tightly links the findings on the molecular level to the level of the biology of the cell line/animal model. Questions and minor revision suggestions for the authors: 1. In the experiments shown in Figure 5, A549 cells were first infected and then treated with lnc-AROD siRNAs. Why not the KD first and then infect the cells? 2. The rationale of why CUEDC2 is selected to be the interest of this study is missing in the manuscript. The authors wrote in the Discussion section about some published functions of CUEDC2, and its association with miR-324-5p. However, it was also mentioned in the Discussion section that CUEDC2 is not the only target of miR-324-5p. I believe the manuscript could benefit from an explanation of the authors' selection of genes of interest. 3. The illustration of the relationship between IAV infection, lnc-AROD abundance, and the associated change of CUEDC2 localization + downstream effect is somewhat misleading in Fig.9 and can be improved. The current expression in the figure is not the best.
Reviewer #3 (Comments for the Author): In their manuscript, Zhang etal employ genome wide RNA-seq profiling in IAV infected or non-infected 549 cell lines and identify several ncRNA that their transcription is changed upon infection. Among their candidates they identify AROD lncRNA and further show that it enhances IAV infection. AROD is defined as important for IAV pathogenesis in vivo and negatively regulates the expression of ISGs. Their data shows that AROD is downregulated in IAV RNA virus-infected cells. Moreover, both in vitro and in vivo AROD overexpression enhances replication of RNA viruses, while no effects were seen on DNA virus replication. They further show that in IAV infected mice, expression of AROD lncRNA via intranasal transduction of AAV, led to slight increased IAV titers and death of infected mice relative to AAV viruses expressing a control GFP gene. Mechanistically they employ detailed in vitro and in vivo functional assays to demonstrate that AROD lncRNA suppresses ISG genes expression, defining AROD as a negative regulator of host innate response. Further analysis shows that AROD-lncRNA acts as a competitor RNA that enhances CUEDC2 expression via sponging miR-324-5p. Both CUEDC2 and miR-324-5p were nicely shown to associate with AROD and further manipulation of AROD lncRNA expression affected their expression. Overall, they conclude that miR-324-5p directly binds AROD and CUEDC2 to suppress the upregulation of CUEDC2 expression enhanced by AROD. Thus AROD regulates CUEDC2 expression by directly targeting miR-324-5p. and AROD induces formation of a complex with miR-324-5p and CUEDC2, targeting miR-324-5p and functioning as a ceRNA to overall suppress ISG expression.
Major remarks 1. It would be beneficiary that the screen for functional ncRNA will be conducted in primary target cells and not in a cell line. At least the validation of expression and effect on viral titer should be repeated in primary cells. 2. The authors show that KD of AROD increases levels of ISGs upon infection (Fig. 5)-No control is available of non-infected cells that either over-express or deplete of AROD incRNA. What is the effect on ISGs expression in control mock cells, where AROD is KD or AROD over expressed; and in infected but not express AROD. 3. Nuclear and cytoplasmic fractions, as well as an in situ hybridization assays detected that the AROD concentration is not significantly decreased in the cytoplasm of cells with or without virus infection. Can the author monitor the levels in nuclear fractions. How does this reside with the overall decrease in AROD levels upon infection?). 4. Can the authors determine the actual copy number of AROD in each of their fractions using quantitative qPCR. 5. In Fig. 1 -please indicate if the enrichment is relative to non-infected cells. One can think to combine Fig 1+2. 6. Fig. 2E -a kinetic of lncRNA levels post infection is appropriate here. 7. Fig.2G -any information on AROD isoforms and the one that is most abundant in humans. 8. Fig3B -Effects are only shown for 16hpi. Can longer time frame be addressed. 9. Fig 3C+D -how significance are the infection differences upon over expressing AROD. It seems very low. Levels of M1 are not changing. 10. Fig 3H. -the effects of AROD silencing on infection is very subtle (x1.5fold). Same for the WB at panel G -differences in NP and M1 upon AROD silencing are very low. Maybe higher effects will be detected at later time points. 11. Fig. 4E -as noted above -the effects of AAV-AROD transduction on IAV titers are extremely low . 12. Fig. 7 -Can the authors also present data on ISG basal levels in non-infected cells (upon miR -mimics or inhibition).

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Reviewer' Comments to Author:
The authors describe a mechanism through which the lnc22 AROD/miR-324-5p/CUEDC2 axis regulates the host innate immune response using 23 influenza A virus as a model. From this article the conclusion is that lnc-AROD is a critical regulator of the host antiviral response via the miR-324-5p/CUEDC2 axis and lnc-AROD functions as competing endogenous RNA.
I due have some questions/suggestions to clarify the methods and about the interpretation of the results: LINE 47= Is there a reference. If so, please add reference in this point.
LINE 275= Further example should be added, together with other references.

Reviewer #1's comments:
The authors describe a mechanism through which the lnc-AROD/miR-324-5p/CUEDC2 axis regulates the host innate immune response using influenza A virus as a model. From this article the conclusion is that lnc-AROD is a critical regulator of the host antiviral response via the miR-324-5p/CUEDC2 axis and lnc-AROD functions as competing endogenous RNA.

Reviewer #2's comments:
In this study, the authors demonstrated an association between the abundance of lnc-AROD and the influenza A virus infection in both human cell lines and in the mouse model. Within the manuscript, the authors claimed the following findings: 1. Lnc-AROD interacts with miR-324-5p, and the interaction is potentially direct; 2. The above interaction leads to a decreased binding of miR-324-5p to the 3' UTR of CUEDC2 mRNA, which then causes an increased abundance of CUEDC2 protein; 3. Lnc-AROD is a critical regulator of the host antiviral response (Lnc-AROD abundance is positively associated with the virus titer post-IAV infection); 4. The antiviral function of lnc-AROD is realized through its direct interaction with miR-324-5p; 5. Through its regulatory effect on CUEDC2 via miR-324-5p, lnc-AROD abundance is negatively associated with the expression level of IFN-β, ISG15, and MxA.
The authors showed experimental evidence to support the above findings from the perspectives of both ex vivo and in vivo. To test the suggested molecular mechanism of action in regulating the innate immune response to IAV by lnc-AROD, the authors conducted both overexpression and knockdown studies using multiple experimental methodologies. They provided solid results to support the conclusion of this manuscript, as well as the suggested working model described in Fig. 9.
In Fig. 3&4, the authors showed that lnc-AROD abundance is associated with the infection rate of IAV, using both KD and overexpression of lnc-AROD. The experimental evidence was collected from both cultured cell lines and mouse models. Through Fig. 6&7, the authors showed evidence to support the hypothesis that lnc-AROD can form a direct interaction with miR-324-5p, and this interaction "competes" the affinity between miR and CUEDC2 mRNA. The authors then suggested that this competition may lead to a modified/regulated expression of downstream genes that play important roles in innate immunity.
Moving on to Fig.8, the authors showed evidence that CUEDC2 itself has an effect on the expression level of the above-mentioned three genes that can be impacted by the level of lnc-AROD.
Moreover, the authors also presented data through the figures to show that viral titer can be impacted by the experimental treatment (such as overexpression of CUEDC2, etc), which tightly links the findings on the molecular level to the level of the biology of the cell line/animal model. 2. The rationale of why CUEDC2 is selected to be the interest of this study is missing in the manuscript. The authors wrote in the Discussion section about some published functions of CUEDC2, and its association with miR-324-5p. However, it was also mentioned in the Discussion section that CUEDC2 is not the only target of miR-324-5p. I believe the manuscript could benefit from an explanation of the authors' selection of genes of interest.
Response: thanks for your comments. In our functional study, we found that lnc-AROD played a negative regulator in innate immunity, and CUEDC2, the target gene of miR324-5p, could also negatively regulates immunity, which is consistent with the function of lnc-AROD. Therefore, we selected the CUEDC2 gene for subsequent research. And the explanation has been added in Discussion section of Revised Manuscript and highlighted in red words (lines 476-478).
3. The illustration of the relationship between IAV infection, lnc-AROD abundance, and the associated change of CUEDC2 localization + downstream effect is somewhat misleading in Fig.9 and can be improved. The current expression in the figure is not the best.
Response: thanks for your comments. According to your comment, we have modified the schematic diagram in Revised Figure 9.

Reviewer #3's comments:
In their manuscript, Zhang etal employ genome wide RNA-seq profiling in IAV infected or non-infected 549 cell lines and identify several ncRNA that their transcription is changed upon infection. Among their candidates they identify AROD lncRNA and further show that it enhances IAV infection. AROD is defined as important for IAV pathogenesis in vivo and negatively regulates the expression of ISGs.
Their data shows that AROD is downregulated in IAV RNA virus-infected cells. Moreover, both in vitro and in vivo AROD over-expression enhances replication of RNA viruses, while no effects were seen on DNA virus replication. They further show that in IAV infected mice, expression of AROD lncRNA via intranasal transduction of AAV, led to slight increased IAV titers and death of infected mice relative to AAV viruses expressing a control GFP gene.
Mechanistically they employ detailed in vitro and in vivo functional assays to demonstrate that AROD lncRNA suppresses ISG genes expression, defining AROD as a negative regulator of host innate response. Further analysis shows that AROD-lncRNA acts as a competitor RNA that enhances CUEDC2 expression via sponging miR-324-5p. Both CUEDC2 and miR-324-5p were nicely shown to associate with AROD and further manipulation of AROD lncRNA expression affected their expression. Overall, they conclude that miR-324-5p directly binds AROD and CUEDC2 to suppress the upregulation of CUEDC2 expression enhanced by AROD. Thus AROD regulates CUEDC2 expression by directly targeting miR-324-5p. and AROD induces formation of a complex with miR-324-5p and CUEDC2, targeting miR-324-5p and functioning as a ceRNA to overall suppress ISG expression.
Major remarks 1. It would be beneficiary that the screen for functional ncRNA will be conducted in primary target cells and not in a cell line. At least the validation of expression and effect on viral titer should be repeated in primary cells.
Response: thanks for your comments. According to your comment, we isolated and cultured the primary lung fibroblasts from the fetal mice (MPF), and electrotransfected lnc-AROD to achieve overexpression of lnc-AROD in primary lung fibroblasts (Please see the following figure A), and then infected H1N1 virus to detect the effect on IAV replication. In viral titer assay, we found that compared with the control group, the H1N1 viral titer was significantly increased in primary mouse lung fibroblasts transfected with pCDH-AROD plasmid by about 10 times (Please see the following figure B). And combined with in vivo experiments in mice (please see Figure 4), the function of lnc-AROD has been determined.  5. In Fig. 1 -please indicate if the enrichment is relative to non-infected cells. One can think to combine Fig 1+2. Response: thanks for your comments. Figure 1 is not the result of enrichment. Figure 1A and B are new lncRNAs identified in infected cells, Figure 1C and D are all lncRNAs identified for classification and chromosome distribution statistics. While, the results in the Heat Maps and Volcano Plot of Figure 2A and B are relative to non-infected cells. Therefore, we prefer to put Figure 1  7. Fig.2G -any information on AROD isoforms and the one that is most abundant in humans.
Response: thanks for your comments. AROD isoforms were not found in our high-throughput sequencing results, and was also proved by RACE experiments. Response: thanks for your comments. According to our grayscale analysis, the expression of M1 and NP were significantly improved in Figure 3C, the data were statistically significant.
In the titer result of Figure 3D, the ordinate is the result of logarithm. Therefore, compared with the control group, overexpression of lnc-AROD can increase the virus titer of H1N1 by about 20 times. We also modified the ordinate character of the virus titer in Figure 3D (Please see Revised Figure 3).
10. Fig 3H. -the effects of AROD silencing on infection is very subtle (x1.5fold). Same for the WB at panel G -differences in NP and M1 upon AROD silencing are very low. Maybe higher effects will be detected at later time points.
Response: thanks for your comments. According to our grayscale analysis, the expression of M1 and NP were significantly decreased in Figure 3G, the data were statistically significant.
In the titer result of Figure 3H, the ordinate is the result of logarithm. Therefore, compared with the control group, knockdown of lnc-AROD can decrease the virus titer of H1N1 by about 100 times. We also modified the ordinate character of the virus titer in Figure 3H (Please see Revised Figure 3).
11. Fig. 4E -as noted above -the effects of AAV-AROD transduction on IAV titers are extremely low.
Response: thanks for your comments. In the titer result of Figure 4F, the ordinate is the result of logarithm. Therefore, IAV virus titer was about 8 times higher in the lungs of AAV-lnc-AROD mice compared to that in the lungs of AAV-GFP mice. We also modified the ordinate character of the virus titer in Figure 4F (Please see Revised Figure 4).
12. Fig. 7 -Can the authors also present data on ISG basal levels in non-infected cells (upon miR -mimics or inhibition).
Response: thanks for your comments. Following your suggestion, we overexpressed miR-324-5p mimcs in uninfected cells to detect the expressions of ISGs. In qPCR assays, we found that compared to the control cells, the expressions of ISGs had no significant change in cells that overexpressed miR-324-5p without virus infection (Please see the following figure A).