Structure of the HIV immature lattice allows for essential lattice remodeling within budded virions

  1. Sikao Guo
  2. Ipsita Saha
  3. Saveez Saffarian
  4. Margaret E Johnson

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    This is a valuable study, carried out in a solid and comprehensive manner. The results advance the understanding of one of the steps of the HIV life cycle, via a better description of the mechanisms underlying Gag-Pol dimerization.

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Abstract

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use spatial stochastic computer simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol is highly likely and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (–12 k B T <∆ G <–8 k B T ) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses.

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  1. Version published to 10.7554/elife.84881 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The authors have approached the study of the mechanism of maturation of retroviruses lattice, where Gag polyprotein is the main component. The Gag polyprotein is common to all retroviruses and makes up most of the observed lattice underlying the virion membrane. Within the lattice, 95% of the monomers are Gag, and 5% are Gag-Pol, which has the 6 domains of Gag followed by protease, reverse transcriptase and integrase domains (coming from Pol) embedded within the same polyprotein. For the maturation and infectivity of HIV retrovirus, the Gag proteins within the immature lattice must be cleaved by the protease formed from a dimer of Gag-Pol. Importantly, the lattice covers only 1/3 to 2/3 of the available space on the membrane. The incompleteness of the lattice results in a periphery of Gag monomers with unfulfilled intermolecular contacts. Recently, the structure of the immature lattice has been partially resolved using sub-tomogram averaging cryotomography (cryET) and it has been shown that the incompleteness of the lattice provides more accessible targets for the protease (Tan A. et al. 2021). Based on these, the authors have wondered: does the incompleteness of the lattice allow for dynamic rearrangements that ensure that protease domains embedded within the lattice can find one another to dimerize and activate? To answer this, they started from experimental cryoET data and used reaction-diffusion simulations of assembled Gag lattices with varying energies and kinetic rates to test how lattice structure and stability can support the dimerization of the Gag-Pols. They found that although they represent only 5% of the monomers that assemble into the lattice, the stochastic assembly ensure that at least a pair of them are adjacent within the lattice. They next showed that if the molecules are distant from one another, they would need to detach, diffuse, and reattach stochastically at the site of another Gag-Pol molecule.

    I consider the work very interesting, which could contribute to a very important aspect of retroviruses maturation such as their infectivity. However, the observations made by the authors do not necessarily answer their initial question which seemed to be focused on studying the possible role of the incompleteness of the lattice on the protease activation rather than the mechanism of Pol activation itself. Maybe this is only a nuance to be polished in the writing.

    The weakness of the work comes from both the fact their entire study has been done by computational methods and the exclusion in their computational approaches of well-known cellular components with a role in retrovirus maturation, which might obey to the fact of keeping their models into the simplest possible since handling atomistic models is already a heavy task. Maybe complementary molecular or structural studies would strengthen their results.

    We appreciate Reviewer #1’s comments and interest in our work. In the revised manuscript, we have clarified our writing to emphasize that our primary goal was to quantitatively interrogate the mechanism by which dimerization of two protease domains can occur, as it is essential for activation of the protease and the subsequent maturation process. We do not address the steps that follow the essential dimerization process. The molecular details concerning how this (dimerized) protease enzyme initiates cleavage and ultimately cleaves the entire lattice off the membrane would be the subject of a separate study (see page 4).

    Regarding the concern about the computational nature of our work, we agree that the model is a (necessarily) simplified representation of the true biological system, and as we elaborate further in the Discussion section, including more components in the model would strengthen connection to experiment, which we plan in future work (see page 26). However, we have not relied solely on our computational results, but made several direct and quantitative comparisons to experimental structural (now!), biochemical, and imaging data. We have also validated the parameters of our model against theory.

    We agree that it would be interesting and worthwhile to make more direct comparisons as well to more molecular models of the immature lattice, particularly in future work with the inclusion of more specific co-factors like IP6, which we discuss in the Discussion section. We show that already in its current form, our model provides new quantitative evidence on the mechanisms that would allow two protease domains to find one another to initiate maturation, in a way that is consistent with structural and biochemical data. (See revisions on page 26 and 28 of the manuscript)

    Reviewer #2 (Public Review):

    Immature lattice assembly remains an arcane topic, and these simulations provide high resolution data such as assembly kinetics and large-scale lattice rearrangement. Further, the authors extend their model to compare directly with experiments, e.g. SNAP-HALO dimerization, which provides a basis to interpret their conclusions. The manuscript is difficult to read, as it is a technical manuscript that overuses jargon; overall, it seems written for a specialized audience. Additionally, there are several aspects of the model design that remain opaque, such as the implicit lipid method and the suppression of multi-site nucleation. Further, analyses such as time auto-correlation and mean first passage time are not given much context by the authors. Altogether, it is the opinion of this reviewer that several revisions to the manuscript should be incorporated to improve clarity and strengthen the significance of the authors' efforts.

    We appreciate the constructive comments from Reviewer #2. We've revised the text in multiple places to minimize the use of technical jargon and provide clearer explanations for specialized terms or concepts. Specifically, we've provided more detailed descriptions of the implicit lipid method and the rationale behind the suppression of multi-site nucleation to help readers better understand our model design. Additionally, we've added more context for the analyses such as time auto-correlation and mean first passage time, describing their significance and relevance to our study.

    Reviewer #3 (Public Review):

    The manuscript concerns the cleavage of the Gag polyprotein lattice from the HIV virion membrane, a key stage in HIV lifecycle, and one that is required for HIV to become infectious. Since cleavage requires homodimerization between the small fraction (5%) of such Gag polyproteins that carry a protease domain, referred to as Gag-Pol, this raises questions regarding how such homodimerization can take place, and whether it can happen on the required timescales, given that Gag-Pol is typically embedded in a lattice that is observed to form one large connected component.

    The authors address these questions in silico, using particle-based reaction diffusion simulations. Such simulations are rigid-body and "structure-resolved" meaning that they rigidly incorporate the geometry of the polyproteins, and their various binding interfaces, based on existing structural data. Other aspects of the simulations are also in-line with available data, including copy numbers, lattice curvature, and dissociation rates. This focused approach is a strength of the work and allows the authors to make credible claims that their simulations have relevance to HIV (as does their commitment to comparison with HALO-SNAP-based measurements of dimerization kinetics as well as iPALM experiments that characterize lattice dynamics).

    A central part of the model is that it allows for the "possibility of imperfect alignment of molecules in the lattice", presumably due to the incompatibility of regular hexagonal tiling and surfaces with non-zero Gaussian curvature, such as a sphere. This is implemented via the ad-hoc imposition of a free-energy penalty when complete hexamers are formed, implying that hexamers are less stable than six ideal bonds. By varying this strain penalty, the authors can change the stability of the lattice independently of individual binding affinities, allowing its use as an effective fitting parameter when comparing to HALO-SNAP data. In the latter case, agreement between simulation and experiment can only be found at moderate levels of lattice stability.

    However, such energetic penalties are present whenever the polyprotein structure must undergo deformations which, on surfaces with nonzero Gaussian curvature, should be the case for partial tilings as well as complete ones (where all six interfaces form bonds). This, therefore, appears to be a weakness of the work. An elastic implementation of polyprotein structure, for example, would permit strain to accumulate (and therefore stresses to propagate) throughout the lattice naturally, irrespective of whether complete hexamers were formed, and might reasonably be expected to impact the likelihood of different lattice structures. Whilst it is not clear how or whether this would lead to qualitatively or quantitatively different results, it is nevertheless worth remarking upon since the authors high-level claim is that lattice structure is an important determinant of the mean-firstpassage times to dimerization.

    Overall, I find this to be a valuable study, carried out in a solid and comprehensive manner. The primary impact of the work appears to be twofold: the unification of different experimental measurements under a single model, and the further identification of the salient parts of that model that most impact biological function. The results advance the understanding of one of the steps of the HIV lifecycle, via a better description of the mechanisms underpinning Gag-Pol dimerization. Notably, the authors stop short of drawing parallels to many related concepts and models in statistical physics, such as those concerning percolation and diffusion limited aggregation as well as the notions of dislocations and defects in crystalline matter on curved surfaces. These might reasonably have provided a basis for better understanding and quantification of the authors' simulations, as well as improving the scope for extensions and conceptual clarity.

    We appreciate the constructive and positive comments from Reviewer #3. We have revised the text and expanded the discussion given the points the reviewer has brought up.

    We have quantified the number and organization of incomplete hexamers or defects in the simulated lattices. This allows us to compare with experimental structures and also quantify how the assembly parameters would impact this organization. As we now remark on pages 10-11, with reversible binding during assembly, we see that fewer defects are present in the lattice, indicating that the hexameric lattice can improve its organization and stability when unbinding reactions can correct for weak contacts in the lattice. We thus speculate that because our lattices are statistically in good agreement with experiment even when binding is irreversible, that the assembly process does not rely on a significant amount of annealing. Otherwise the lattice structures would be more ideal.

    We further discuss on pages 27 that a model that also incorporates forces to control interactions would be important to measure the mechanical stability of the lattice, particularly as it couples to membrane bending. However, models that naturally incorporate forces (via interaction potentials) can be difficult to tune with respect to their kinetics and free energies, which are nontrivial to calculate, unlike our modeling approach here.

    In these sections we have now drawn parallels, as suggested by the reviewer, to the literature on defects in crystalline matter on curved surfaces, and their possible consequences for the mechanics of the lattice (Ref 41 Negri et al, Deformation and failure of curved colloidal crystal shells. Proc Natl Acad Sci U S A, 2015. 112(47)) (Ref 59 Zandi, R. and D. Reguera, Mechanical properties of viral capsids. Phys Rev E Stat Nonlin Soft Matter Phys, 2005. 72(2 Pt 1): p. 021917.)

    We did not include a connection to Diffusion-limited aggregation, as this process seems to result in fractal-like structures that lack the specific hexagonal order of the Gag protein lattice. The proteins impose a significant orientational order on the assembly process that makes growth significantly more compact, at least under the assembly conditions we used. Even for our fastest rates, the process is still only moderately diffusion influenced (ka <<109M-1s-1), with typically multiple collisions needed before succesful binding occurs, consistent with most protein-protein interactions.

  3. eLife

    eLife assessment

    This is a valuable study, carried out in a solid and comprehensive manner. The results advance the understanding of one of the steps of the HIV life cycle, via a better description of the mechanisms underlying Gag-Pol dimerization.

  4. eLife

    Reviewer #1 (Public Review):

    The authors have approached the study of the mechanism of maturation of retroviruses lattice, where Gag polyprotein is the main component. The Gag polyprotein is common to all retroviruses and makes up most of the observed lattice underlying the virion membrane. Within the lattice, 95% of the monomers are Gag, and 5% are Gag-Pol, which has the 6 domains of Gag followed by protease, reverse transcriptase and integrase domains (coming from Pol) embedded within the same polyprotein. For the maturation and infectivity of HIV retrovirus, the Gag proteins within the immature lattice must be cleaved by the protease formed from a dimer of Gag-Pol. Importantly, the lattice covers only 1/3 to 2/3 of the available space on the membrane. The incompleteness of the lattice results in a periphery of Gag monomers with unfulfilled intermolecular contacts. Recently, the structure of the immature lattice has been partially resolved using sub-tomogram averaging cryotomography (cryET) and it has been shown that the incompleteness of the lattice provides more accessible targets for the protease (Tan A. et al. 2021). Based on these, the authors have wondered: does the incompleteness of the lattice allow for dynamic rearrangements that ensure that protease domains embedded within the lattice can find one another to dimerize and activate? To answer this, they started from experimental cryoET data and used reaction-diffusion simulations of assembled Gag lattices with varying energies and kinetic rates to test how lattice structure and stability can support the dimerization of the Gag-Pols. They found that although they represent only 5% of the monomers that assemble into the lattice, the stochastic assembly ensure that at least a pair of them are adjacent within the lattice. They next showed that if the molecules are distant from one another, they would need to detach, diffuse, and reattach stochastically at the site of another Gag-Pol molecule.

    I consider the work very interesting, which could contribute to a very important aspect of retroviruses maturation such as their infectivity. However, the observations made by the authors do not necessarily answer their initial question which seemed to be focused on studying the possible role of the incompleteness of the lattice on the protease activation rather than the mechanism of Pol activation itself. Maybe this is only a nuance to be polished in the writing.

    The weakness of the work comes from both the fact their entire study has been done by computational methods and the exclusion in their computational approaches of well-known cellular components with a role in retrovirus maturation, which might obey to the fact of keeping their models into the simplest possible since handling atomistic models is already a heavy task. Maybe complementary molecular or structural studies would strengthen their results.

  5. eLife

    Reviewer #2 (Public Review):

    Immature lattice assembly remains an arcane topic, and these simulations provide high resolution data such as assembly kinetics and large-scale lattice rearrangement. Further, the authors extend their model to compare directly with experiments, e.g. SNAP-HALO dimerization, which provides a basis to interpret their conclusions. The manuscript is difficult to read, as it is a technical manuscript that overuses jargon; overall, it seems written for a specialized audience. Additionally, there are several aspects of the model design that remain opaque, such as the implicit lipid method and the suppression of multi-site nucleation. Further, analyses such as time auto-correlation and mean first passage time are not given much context by the authors. Altogether, it is the opinion of this reviewer that several revisions to the manuscript should be incorporated to improve clarity and strengthen the significance of the authors' efforts.

  6. eLife

    Reviewer #3 (Public Review):

    The manuscript concerns the cleavage of the Gag polyprotein lattice from the HIV virion membrane, a key stage in HIV lifecycle, and one that is required for HIV to become infectious. Since cleavage requires homodimerization between the small fraction (5%) of such Gag polyproteins that carry a protease domain, referred to as Gag-Pol, this raises questions regarding how such homodimerization can take place, and whether it can happen on the required timescales, given that Gag-Pol is typically embedded in a lattice that is observed to form one large connected component.

    The authors address these questions in silico, using particle-based reaction diffusion simulations. Such simulations are rigid-body and "structure-resolved" meaning that they rigidly incorporate the geometry of the polyproteins, and their various binding interfaces, based on existing structural data. Other aspects of the simulations are also in-line with available data, including copy numbers, lattice curvature, and dissociation rates. This focused approach is a strength of the work and allows the authors to make credible claims that their simulations have relevance to HIV (as does their commitment to comparison with HALO-SNAP-based measurements of dimerization kinetics as well as iPALM experiments that characterize lattice dynamics).

    A central part of the model is that it allows for the "possibility of imperfect alignment of molecules in the lattice", presumably due to the incompatibility of regular hexagonal tiling and surfaces with non-zero Gaussian curvature, such as a sphere. This is implemented via the ad-hoc imposition of a free-energy penalty when complete hexamers are formed, implying that hexamers are less stable than six ideal bonds. By varying this strain penalty, the authors can change the stability of the lattice independently of individual binding affinities, allowing its use as an effective fitting parameter when comparing to HALO-SNAP data. In the latter case, agreement between simulation and experiment can only be found at moderate levels of lattice stability.

    However, such energetic penalties are present whenever the polyprotein structure must undergo deformations which, on surfaces with nonzero Gaussian curvature, should be the case for partial tilings as well as complete ones (where all six interfaces form bonds). This, therefore, appears to be a weakness of the work. An elastic implementation of polyprotein structure, for example, would permit strain to accumulate (and therefore stresses to propagate) throughout the lattice naturally, irrespective of whether complete hexamers were formed, and might reasonably be expected to impact the likelihood of different lattice structures. Whilst it is not clear how or whether this would lead to qualitatively or quantitatively different results, it is nevertheless worth remarking upon since the authors high-level claim is that lattice structure is an important determinant of the mean-first-passage times to dimerization.

    Overall, I find this to be a valuable study, carried out in a solid and comprehensive manner. The primary impact of the work appears to be twofold: the unification of different experimental measurements under a single model, and the further identification of the salient parts of that model that most impact biological function. The results advance the understanding of one of the steps of the HIV lifecycle, via a better description of the mechanisms underpinning Gag-Pol dimerization. Notably, the authors stop short of drawing parallels to many related concepts and models in statistical physics, such as those concerning percolation and diffusion limited aggregation as well as the notions of dislocations and defects in crystalline matter on curved surfaces. These might reasonably have provided a basis for better understanding and quantification of the authors' simulations, as well as improving the scope for extensions and conceptual clarity.

  7. Version published to 10.1101/2022.11.21.517392v1 on bioRxiv