Decision letter | The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA

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The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA

Decision letter

Affiliation details

University of California, San Francisco, United States
Wesley I Sundquist, Reviewing editor, University of Utah, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “HIV utilizes a Crm1 dimer to promote RNA nuclear export” for consideration at eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor), Wes Sundquist (Reviewing editor), and 2 reviewers, one of whom, Jamie Williamson, has agreed to share his identity.

The authors describe the reconstitution from pure recombinant components and an EM reconstruction of the HIV-1 RNA export complex: i.e., a complex formed between the HIV-1 RRE and Rev, Ran(GTP), and the human exportin, Crm1. The authors clearly demonstrate that all of these components are required for efficient complex formation and they use a mutant Rev to show that the necessary interaction between Crm1 and Rev NES is indeed taking place. They then go on to present the surprising, but compelling argument that there are two copies of hCrm1 in the complex, rather than the expected single copy, even though Crm1 is monomeric in solution. The presence of a Crm1 dimer is shown both biochemically and by EM. In the EM work, a nominal ∼30 Å resolution reconstruction is generated by negative-stain single-particle electron microscopy, using the random conical tilt method to achieve a suitable distribution of viewing angles. The authors can fit a dimer extracted from the lattice of the hCrm1 crystal structure into their reconstruction, even though the intermolecular lattice contact had previously been assumed to be a crystallization artifact. Nicely, the human Crm1 dimer interface also appears to explain, at least in part, why HIV-1 Rev uses human Crm1 better than murine Crm1 (where the dimer interface is missing). The RRE-Rev complex binds more tightly and with greater cooperativity to the human Crm1-RanGTP complex vs. the murine complex (although the differences are modest), and residues that appear to dictate the species specificity of HIV-1 Rev map to the hCrm1 crystallographic dimer interface. Finally, the authors propose a model for the overall architecture of the HIV-1 export complex and posit that the spacing between the Rev binding sites on the Crm1 dimer are optimally distanced to recognized the C-terminal tails of the two terminal Revs in a hexameric complex.

Despite the relatively low resolution of the EM reconstruction, this paper is important because it reveals a new functional oligomeric state for hCrm1 (which may also be used in the export of larger host RNA complexes) and because it provides a satisfying molecular explanation for the well-known inability of HIV-1 Rev to utilize murine Crm1 efficiently. Although Crm1 has been previously characterized as binding other export cargoes as a monomer, the authors argue that Rev-RRE stabilizes the formation of a Crm1 dimer and that the dimer requirement reflects the greater size of this export cargo. The basic observations presented in this paper are an important advance in thinking about the Rev-RRE-Crm1 interactions, and how the HIV (and other) export complexes are formed.

Major issue (which must be addressed by the authors):

No additional experiments need to be done, but the authors should back off from their overly detailed model for the Rev/RRE component of the export complex because the information going into the modeling exercise is not sufficiently secure or precise to support the model. Issues limiting the precision of the reconstruction include: 1) The reconstruction is in negative stain and is at low resolution. 2) There seems to be a large amount of density missing from the complex. Even given that RNA does not stain well during negative staining, Rev-GB1 should stain well, and Rev could be seen as four round spheres in a previous reconstruction of Rev in complex with the RRE. The authors cite this paper, but do not compare that structure with the present structure, nor do they clearly state why there would be such a difference between the two (namely the complete lack of Rev-density in the current data). Furthermore, the Rev-RRE subcomplex adds up to about 75 kDa and should show up more strongly in the class averages than it does. This raises the concern that the suggested stoichiometry may be incorrect and/or that the averages acquired may include breakdown products and the preps used for EM may not have been exactly the same as those analyzed biochemically. 3) The (red) difference density that is shown in Figure 3, panels (a) and (b) is shallow and smeared a long way over the side of the complex and is hard to reconcile with the difference densities seen in the class averages shown in panel (c). 4) The interpretation of the difference density fails to mention the possibility that ordering of the Rev CTD may be taking place.

Similarly, the modeling has not (and probably cannot be) performed rigorously given the paucity of solid biochemical and structural information on the stoichiometry and orientation of the Rev-RRE complex. Issues include: 1) A lack of justification for the assumed jellyfish model and Rev stoichiometry in the context of the Crm1:RRE complex. 2) Questions surrounding the Rev subunits whose NES elements do not engage Crm1. The model suggests that six Rev monomers are correctly spaced to allow the terminal Rev monomers to interact with their binding sites on Crm1. However, it is not entirely clear that, based on the data in the accompanying crystal structure of a Rev dimer:RRE complex, the jellyfish model is the appropriate complex to consider. Furthermore, nothing is said of what happens with the intervening four monomeric tails. These would seem to be steric hindrances in the Crm1-Rev interaction, as it is not clear that the space between Rev and Crm-1 would allow nearly 23 kD of unstructured protein mass.

In essence, the reconstruction and the difference density reveal only the general site where the Rev-RRE complex binds the Crm1 dimer. This should be noted and is of some interest because it differs from the crystallographically-defined binding site for Snurportin1. However, we suggest backing off on the detailed interpretation of the complex structure (except for the Crm1 fitting, which appears solid) and omitting Figure 3d because there is no experimental evidence either here or in the companion paper for a Rev hexamer. Alternatively, the authors could consider obtaining a higher resolution cryo-EM reconstruction and/or providing much stronger biochemical evidence for the stoichiometry and structure of the bound Rev-RRE complex.

Other issues for the authors' consideration:

1) Abstract “Here we present the first view of the entire virus-host complex by single-particle electron microscopy.” Suggest rewording as “the assembled complex of known export factors”, explicitly include “negative stain” in the “single particle electron microscopy” (otherwise, people tend to assume that cryo-EM is meant) and to allow the possibility that (currently unknown) factors may be present in the functional complex.

2) Second paragraph of Introduction: “Here we report the single-particle electron microscopy (EM) structure of the Rev-RRE/Crm1-RanGTP export complex.” Caveat. While assembly of the complex is a notable achievement on its own, the wording here belies the reality that the difference density does not reasonably or unambiguously accommodate Rev or the RRE.

3) The text explanation of the experiment being performed in Figure 1a is incomplete/confusing. The experimental result is clear enough, but the authors should clarify the description of the source of the different samples and not force the reader to go through the experimental methods to understand what was done.

4) Figure 2 and Figure 2–figure supplement 2. A) Clarify that the reconstruction in Figure 2a is of the full Rev-RRE-Crm1-RanGTP complex. The figure legend needs to reflect this because otherwise it appears to be a reconstruction of a Crm1 dimer in the absence of Rev-RRE, which would contradict the main argument of the paper. B) It is difficult to believe the correlation coefficients of 0.97 and 0.98 for the claimed fits, which imply that the fits are essentially perfect. This is not the impression one gets from visual appraisal in which there are sizable unoccupied spaces inside the EM envelope. How were the volumes compared normalized? Are their densities sitting on top of large and equal constant densities? C) The fits shown in Figure 2-figure supplement 2 appear convincing, but the authors should provide statistical evidence that the crystallographic human Crm1 dimer structure does not fit well into the mCrm1/Ran(GTP) complex and that the separated monomers from the fit of the mCrm1/Ran(GTP) cannot be well accommodated by the hCrm1 dimer density.

5) “…by single-particle negative-stain EM using random conical tilt…”: Give some brief general account of how random conical tilt works and why it is useful, and give a couple of references. The vast majority of potential readers will never have heard of RCT and the Methods section is not much help.

Similarly, the authors should add a sentence or two that helps the general reader to understand that Ran(GTP) binds on the interior of the Crm1 solenoid and stabilizes a strained conformation that creates the hydrophobic NES binding cleft (and also comment on whether the Ran(GTP) complex is required to explain the observed EM density).

6) The authors state: “Rev and the RRE are unambiguously positioned on one side of the Crm1 dimer, with Rev close to Crm1 and the RRE slightly further away” Where is the evidence for the latter part of that sentence?

7) A crystal structure of the RanGTP-CRM1 complex bound to the Rev NES has been reported (Güttler et al., 2010). Why hasn't the footprint of this Rev NES-Crm1 complex been compared with the data presented herein?

8) An example of the overly detailed modeling/interpretation occurs in the Results section: “Given the flexible nature of the Rev carboxy-terminus, there may be some variation in this distance that allows an NES from a neighboring subunit to be utilized, potentially increasing avidity from a high local concentration of NESs”. The authors' idea seems to be that avidity effects are (relatively) low because the NESs from the terminal Rev molecules in the hexamer are normally used and that avidity effects would be increased if NESs from non-terminal Rev molecules were used. This is an overly detailed and relatively weak argument: why not just leave in the final two sentences, which seem to cover how RNA-dependent remodeling of the REV assembly could modulate Crm1 binding affinity?

9) The authors should elaborate on the meaning of the observed Hill coefficients for murine and human Crm1 binding to the Rev-RRE complex (2.12 and 2.37, respectively). Unless we're missing something, the Hill coefficient should not actually exceed 2 in either case (i.e., the number of Crm1 molecules that bind the Rev-RRE complex). Also, why do the authors think that the mCrm1 molecules bind cooperatively to the Rev-RRE complex if they don't actually touch one another? Finally, it is difficult to believe that the Hill coefficients of 2.37 +/- 0.13 and 2.12 +/- 0.25 are really different at the 0.0001 confidence level. Please double check this.

10) It is a bit surprising that mCrm1 does not dimerize if this oligomeric state is used in the export of some human RNA complexes, and this issue probably merits comment.

DOI: http://dx.doi.org/10.7554/eLife.04121.015