Initiation of HIV-1 Gag lattice assembly is required for recognition of the viral genome packaging signal
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eLife assessment
This work presents valuable findings that advance our understanding of the roles of the CA domain in specific binding of HIV-1 Gag to the viral genomic RNA. The compelling evidence obtained using the modified CLIP-seq and chemical crosslinking approaches support the authors' conclusion that the initial Gag lattice formation mediated by CA is essential for Gag recognition of the 5' Ψ sequence. This work will be of interest to virologists working on gRNA packaging of not only HIV-1 but also other RNA viruses.
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Abstract
The encapsidation of HIV-1 gRNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal (Ψ) at the 5’ end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-Ψ encounter remain uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to Ψ recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to Ψ in living cells, whereas full-length Gag and a CANC subdomain bind to Ψ with high specificity. Perturbation of the Ψ RNA structure or NC zinc fingers affected CANC:Ψ binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer, or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice were unable to specifically bind to Ψ. Artificially multimerized NC domains did not specifically bind Ψ. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in Ψ binding and second-site revertant mutants that restored CA assembly also restored specific binding to Ψ. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and Ψ in cells.
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eLife assessment
This work presents valuable findings that advance our understanding of the roles of the CA domain in specific binding of HIV-1 Gag to the viral genomic RNA. The compelling evidence obtained using the modified CLIP-seq and chemical crosslinking approaches support the authors' conclusion that the initial Gag lattice formation mediated by CA is essential for Gag recognition of the 5' Ψ sequence. This work will be of interest to virologists working on gRNA packaging of not only HIV-1 but also other RNA viruses.
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Reviewer #1 (Public Review):
In this paper Lei et al analyzed the interaction between HIV-1 Gag and the viral RNA packaging signal Psi in living cells using the CLIP-seq method. The authors convincingly showed that NC alone is not sufficient to bind specifically to Psi sequence, while CANC does. They further showed that CANC mutants that are deficient in CA multimerization failed to bind specifically to the Psi sequence. The results indicate that correct assembly of Gag is required for specific binding of the protein to the Psi sequence.
Most of the data are convincing and support the conclusions. My only concern is that the authors analyzed the binding of some CA mutant proteins to the Psi sequence, but it is not so clear whether the specific binding of these mutants would lead to effective packaging of the RNA into virions. Measuring …
Reviewer #1 (Public Review):
In this paper Lei et al analyzed the interaction between HIV-1 Gag and the viral RNA packaging signal Psi in living cells using the CLIP-seq method. The authors convincingly showed that NC alone is not sufficient to bind specifically to Psi sequence, while CANC does. They further showed that CANC mutants that are deficient in CA multimerization failed to bind specifically to the Psi sequence. The results indicate that correct assembly of Gag is required for specific binding of the protein to the Psi sequence.
Most of the data are convincing and support the conclusions. My only concern is that the authors analyzed the binding of some CA mutant proteins to the Psi sequence, but it is not so clear whether the specific binding of these mutants would lead to effective packaging of the RNA into virions. Measuring RNA/Gag ratios of some of the mutants in Figure 4 might help to address this concern.
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Reviewer #2 (Public Review):
To advance the understanding of the initial events in recognition of HIV-1 genome by the viral structural protein Gag, in this study, the authors examined the involvement of the CA domain in the specific interaction between Gag and the viral genomic RNA. Previous studies including a study from the same group (Kutluay et al 2010) showed that the CA C-terminal domain plays a role in Gag binding to viral genomic RNA. In the current study, they analyzed a panel of CA mutants using a modified PAR-CLIP RNA sequencing, which allows identification of Gag binding sites in the viral genome, and a chemical crosslinking approach, which allows assessment of the multimerization status of Gag in cells. They found that substitutions of CA residues at the CA dimer, trimer, or hexamer interfaces, which reduce Gag …
Reviewer #2 (Public Review):
To advance the understanding of the initial events in recognition of HIV-1 genome by the viral structural protein Gag, in this study, the authors examined the involvement of the CA domain in the specific interaction between Gag and the viral genomic RNA. Previous studies including a study from the same group (Kutluay et al 2010) showed that the CA C-terminal domain plays a role in Gag binding to viral genomic RNA. In the current study, they analyzed a panel of CA mutants using a modified PAR-CLIP RNA sequencing, which allows identification of Gag binding sites in the viral genome, and a chemical crosslinking approach, which allows assessment of the multimerization status of Gag in cells. They found that substitutions of CA residues at the CA dimer, trimer, or hexamer interfaces, which reduce Gag multimerization as expected, also reduce the Ψ sequence-specific viral RNA binding, whereas substitutions elsewhere in CA have no impact. They further found that substitutions of the Lys residues important for IP6 binding, which disrupt Gag lattice formation, reduce the Ψ-specific RNA binding, whereas a second-site mutation that restores virus assembly in these Lys substitution mutants restores the RNA binding. These results strongly support the authors' conclusion that Gag lattice formation driven by CA plays an important role in NC-mediated recognition of the Ψ sequence.
The strengths of the work include the application of the modified PAR-CLIP method to the analysis of a large panel of CANC constructs. This provided the detailed information on the specific molecular features in CA required for interactions between Gag and the Ψ sequence, which was not obtainable in the previous studies. The absence of the MA domain in these constructs allowed the authors to focus on the cytoplasmic interactions. The data obtained with oligomer-forming NC constructs and CANC constructs that differ in the IP6 dependence also add support to the authors conclusion that CA-mediated lattice formation of CANC and not just NC oligomerization plays a key role in Gag-vRNA binding. Overall, the data support the conclusion that the ability to form the CANC lattice is essential for the initial NC-vRNA interaction.
The only notable weakness is that previous work by this group and others have already shown that CA and/or its interaction interfaces plays an important role in the Gag-vRNA interaction. Therefore, the current work can be regarded as a refinement of the previously presented concept rather than a conceptual breakthrough. Nonetheless, these mechanistic details are likely to help the retrovirology community gain a clearer grasp of the early steps of infectious particle formation.
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Reviewer #3 (Public Review):
The authors' aim was to examine the early stages of the HIV-1 packaging process inside cells, with specific focus upon how the Gag protein and its cognate domains mediate the initial interaction with the packaging signal on the genomic RNA. The technique that has generated the majority of results in the paper is a modified version of CLIP. The authors have achieved this aim well, with data that clearly support the importance of Capsid, as well as the importance of two different aspects of RNA structure, the IP6 binding site, and various sites that help to form the dimer, trimer and hexamer interfaces on Gag. The major conclusions of the paper, that an immature Gag lattice is needed to form, that NC alone is insufficient to mediate specific recognition of the packaging signal within cells, and that various …
Reviewer #3 (Public Review):
The authors' aim was to examine the early stages of the HIV-1 packaging process inside cells, with specific focus upon how the Gag protein and its cognate domains mediate the initial interaction with the packaging signal on the genomic RNA. The technique that has generated the majority of results in the paper is a modified version of CLIP. The authors have achieved this aim well, with data that clearly support the importance of Capsid, as well as the importance of two different aspects of RNA structure, the IP6 binding site, and various sites that help to form the dimer, trimer and hexamer interfaces on Gag. The major conclusions of the paper, that an immature Gag lattice is needed to form, that NC alone is insufficient to mediate specific recognition of the packaging signal within cells, and that various aspects of Capsid are necessary, are clearly supported by the data.
A particular strength of the paper is the way in which the viral protein and RNA are expressed within cells - these derive from the same construct, which is essentially the proviral genome with mutations to enable the authors to study the various truncations/mutations of Gag and/or the RNA structure. The authors could instead have transfected separate packaging signal/gRNA and viral protein plasmids, but in ensuring that the viral proteins are translated from the same RNA molecule that can also be packaged, they recapitulate the native viral situation in a state of the art experimental form. This is important in terms of the conclusions they can draw, because although HIV-1 can co-package some other lentiviruses, and HIV-1 packaging can occur in trans (ie where 2 gRNA molecules are packaged by molecules of Gag that have not been translated from them), the experiments determining copackaging ability are sometimes not performed in a competitive or limiting system, so it is difficult to say whether there is indeed some remaining importance of co-translational packaging in the very early stages of HIV-1 Gag-psi recognition. Expression of gRNA and protein from the same construct also ensures a balance in stoichiometry within the cytoplasm that is representative of a native infection.
The weakness within the paper is the lack of consideration of how Gag concentration within the cytosol may affects its binding kinetics, both with itself and with the RNA. The CLIP experiments are internally controlled in that they measure binding to the packaging signal relative to the rest of the genome; however, the authors do not appear to have checked that all constructs were expressing at roughly equivalent amounts. This is especially important when interpreting data from a protein such as Gag, which undergoes very complex multimerization, and when considering that the RNA also multimerizes. Both of these multi-step events may alter according to the actual concentrations of both Gag and RNA, and not just the stoichiometric ratio of the two. Some of the data that are needed to provide this evidence are present within the paper already, as western blots analysing multimerization of Capsid mutants, and look to broadly support the expression of the constructs at similar levels. More consideration of this point would strengthen the paper.
The authors place their findings in the context of the field very well. They appear to have considered multiple lines of evidence and to have accounted broadly for previous work done. I do find the discussion of Capsid mutants, and the dimer, trimer and hexamer interfaces quite protein-centric though. I wonder whether there might be a larger role for the RNA structure and structural changes in bringing together the precise Gag lattice structure in some sort of step-wise fashion.
Overall, the manuscript is of great value to the retroviral research community, as it provides data from a highly relevant biological setting. Such data has largely been lacking within the field.
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