Mapping HIV-1 RNA Structure, Homodimers, Long-Range Interactions and persistent domains by HiCapR
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eLife Assessment
This manuscript focuses on the identification of RNA crosslinks within the HIV RNA genome under different conditions i.e. in infected cells and in virions using a new method called HiCapR. These cross-links reveal long-range interactions that can be used to determine the structural arrangement of the viral RNA, providing useful data that show differences in the genomic organization in different conditions. The data analysis, however, is incomplete and based on extensive computational analysis from a limited number of datasets, which are in need of experimental validation.
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Abstract
Human Immunodeficiency Virus (HIV) persists as a leading global health issue. A significant knowledge gap exists in our understanding of long-range interactions of the HIV-1 RNA genome. To bridge this gap, we introduce HiCapR, incorporating a psoralen crosslinking RNA proximity ligation and post-library hybridization for capturing HIV RNA:RNA interactions.
Leveraging HiCapR, we confirm the presence of stem structures in the key regions, such as the 5’-UTR and RRE stems, and dimer sites in 5’-UTR region, which is responsible for HIV packaging. Importantly, we reveal multiple previously unknown homodimers along the HIV genome, which may have important implications for viral RNA splicing and packaging processes. Also, we uncover a wealth of unprecedented long-range interactions, particularly within the 5’-UTR of infected cells.
Intriguingly, our findings indicate a pronounced reduction in long-range RNA:RNA interactions, signifying a transition from a state of abundant interactions, hence a relative loose state within infected cells to a condensed structure within virions. Concurrently, we have demonstrated the presence of stable genomic domains within virions that are instrumental in the dimerization process. These domains are preserved throughout the packaging process.
Our findings shed light on the functional significance of RNA organization, including stable and persistent genomic domains, homodimerization, and long-range RNA:RNA interactions, in the splicing, packaging as well as assembly of HIV.
Highlights
HiCapR is a new proximity ligation method for mapping RNA structures and homodimers in the HIV genome with sufficient reliability and efficiency.
Multiple homodimers were discovered along the genome, with potential implications for splicing and packaging processes.
Long-range RNA:RNA interactions are abundant in infected cells but significantly reduced in virions.
Stable genomic domains encluding homodimer sites are persistent in virions and are involved in dimerization.
Article activity feed
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eLife Assessment
This manuscript focuses on the identification of RNA crosslinks within the HIV RNA genome under different conditions i.e. in infected cells and in virions using a new method called HiCapR. These cross-links reveal long-range interactions that can be used to determine the structural arrangement of the viral RNA, providing useful data that show differences in the genomic organization in different conditions. The data analysis, however, is incomplete and based on extensive computational analysis from a limited number of datasets, which are in need of experimental validation.
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Reviewer #1 (Public review):
This paper focuses on secondary structure and homodimers in the HIV genome. The authors introduce a new method called HiCapR which reveals secondary structure, homodimer, and long-range interactions in the HIV genome. The experimental design and data analysis are well-documented and statistically sound. However, the manuscript could be further improved in the following aspects.
Major comments:
(1) Please give the full name of an abbreviation the first time it appears in the paper, for example, in L37, "5' UTR" "RRE".
(2) The introduction could be strengthened by discussing the limitations of existing methods for studying HIV RNA structures and interactions and highlighting the specific advantages of the HiCapR method.
(3) Please reorganize Results Part 1.
(4) Is there any reason that the authors mention …
Reviewer #1 (Public review):
This paper focuses on secondary structure and homodimers in the HIV genome. The authors introduce a new method called HiCapR which reveals secondary structure, homodimer, and long-range interactions in the HIV genome. The experimental design and data analysis are well-documented and statistically sound. However, the manuscript could be further improved in the following aspects.
Major comments:
(1) Please give the full name of an abbreviation the first time it appears in the paper, for example, in L37, "5' UTR" "RRE".
(2) The introduction could be strengthened by discussing the limitations of existing methods for studying HIV RNA structures and interactions and highlighting the specific advantages of the HiCapR method.
(3) Please reorganize Results Part 1.
(4) Is there any reason that the authors mention "genome structure of SARS-CoV-2" in L95?
(5) L102: Please clarify the purpose of comparing "NL4-3" and "GX2005002." Additionally, could you explain what NL4-3 and GX2005002 are? The connection between NL4-3, GX2005002, and HIV appears to be missing.
(6) Figure 1A is not able to clearly present the innovation point of HiCapR.
(7) Please compare the contact metrics detected by HiCapR and current techniques like SHAPE on the local interactions to assess the accuracy of HiCapR in capturing local RNA interactions relative to established methods.
(8) The paper needs further language editing.
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Reviewer #2 (Public review):
Summary:
In the manuscript "Mapping HIV-1 RNA Structure, Homodimers, Long-Range Interactions and 1 persistent domains by HiCapR" Zhang et al report results from an omics-type approach to mapping RNA crosslinks within the HIV RNA genome under different conditions i.e. in infected cells and in virions. Reportedly, they used a previously published method which, in the present case, was improved for application to RNAs of low abundance.
Their claims include the detection of numerous long-range interactions, some of which differ between cellular and virion RNA. Further claims concern the detection and analysis of homodimers.
Strengths:
(1) The method developed here works with extremely little viral RNA input and allows for the comparison of RNA from infected cells versus virions.
(2) The findings, if validated …
Reviewer #2 (Public review):
Summary:
In the manuscript "Mapping HIV-1 RNA Structure, Homodimers, Long-Range Interactions and 1 persistent domains by HiCapR" Zhang et al report results from an omics-type approach to mapping RNA crosslinks within the HIV RNA genome under different conditions i.e. in infected cells and in virions. Reportedly, they used a previously published method which, in the present case, was improved for application to RNAs of low abundance.
Their claims include the detection of numerous long-range interactions, some of which differ between cellular and virion RNA. Further claims concern the detection and analysis of homodimers.
Strengths:
(1) The method developed here works with extremely little viral RNA input and allows for the comparison of RNA from infected cells versus virions.
(2) The findings, if validated properly, are certainly interesting to the community.
Weaknesses:
(1) On the communication level, the present version of the manuscript suffers from a number of shortcomings. I may be insufficiently familiar with habits in this community, but for RNA afficionados just a little bit outside of the viral-RNA-X-link community, the original method (reference 22) and the presumed improvement here are far too little explained, namely in something like three lines (98-100). This is not at all conducive to further reading.
(2) Experimentally, the manuscript seems to be based on a single biological replicate, so there is strong concern about reproducibility.
(3) The authors perform an extensive computational analysis from a limited number of datasets, which are in thorough need of experimental validation.
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