A coarse-grain computational method integrates biophysical and structural data to generate models of HIV-1 genomic RNA, nucleocapsid and integrase condensed into a mature ribonucleoprotein complex. Several hypotheses for the initial structure of the genomic RNA and oligomeric state of integrase are tested. In these models, integrase interaction captures features of the relative distribution of gRNA in the immature virion and increases the size of the RNP globule, and exclusion of nucleocapsid from regions with RNA secondary structure drives an asymmetric placement of the dimerized 5’UTR at the surface of the RNP globule.
Electron microscopy of mature HIV-1 shows a condensed ribonucleoprotein (RNP) complex [1] packaged within the cone-shaped capsid, which is thought to include genomic RNA, nucleocapsid, integrase, transfer RNA, reverse transcriptase, and other components [2]. It is formed through a complex, multistep process where genomic RNA (gRNA) associates with a lattice of Gag polyproteins at the cell surface, which buds from the surface to form an immature virion, followed by proteolytic cleavage of Gag into capsid, nucleocapsid, integrase and other viral proteins, and finally condensation and encapsidation of the mature RNP within the capsid (Fig 1). Understanding of the supramolecular and molecular details of this RNP is important, since it must undergo large structural transitions over the course of the viral life cycle, including playing a central role in assembly of Gag proteins into new viruses and transition from single-stranded RNA to double-stranded DNA during reverse transcription. The HIV-1 RNP has been studied by many complementary experimental methods. As revealed by SHAPE and other methods, HIV-1 genomic RNA dimerizes and has an extensive secondary structure [4,5], including a well-documented structure at the 5’ untranslated region (5’UTR) [6]. Nucleocapsid coats the gRNA at a density of about 1 nucleocapsid per 11–12 nucleotides and plays key roles as a chaperone in reverse transcription and other processes [7]. A recent study took advantage of the extreme radiation sensitivity of nucleocapsid, which causes formation of bubbles in tomograms, to localize nucleocapsid in the RNP and further show a preference for localization in the dense condensate at the large end of the capsid [8]. Studies on the mode of action of ALLINI compounds (allosteric integrase inhibitors) have revealed that integrase is also essential for maturation of the RNP, and crosslinks the RNA in the mature virion [9]. As part of our ongoing work to study the mesoscale structure of HIV virions at various stages of the viral life cycle, we have developed coarse-grain models of the mature HIV-1 RNP to explore its formation and characteristics, integrating experimental results from electron microscopy, structural biology, CLIP-Seq and SHAPE. Coarse-grain models have been instrumental in understanding of mesoscale-level protein interactions in HIV structure and maturation, including models of the structure of immature virion [10] and formation of the cone-shape capsid [11,12]. In particular, these studies reveal the process whereby gag protein in immature virions assembles into an imperfect hexagonal lattice. CryoEM studies have further revealed that this hexagonal lattice covers roughly 2/3 of the inner surface of the immature virion, with scattered defects [13]. In the work reported here, we interpret these results with a quasisymmetrical model of gag in immature virions, where the defects correspond to missing gag hexamers at the pentagonal sites in the quasisymmetrical lattice. We then generate models of the mature RNP that explore several alternatives for how the gRNA is deposited onto this immature gag lattice, and how much of this immature virion structure is retained when the RNP matures. Our RNP models include three elements: two copies of 9 kb gRNA, 2000 nucleocapsid proteins, and 140 integrase subunits. The coarse-grain method begins with several assumptions for the structure of the genome within the immature virion, folds the 5’UTR based on experimentally-determined base-pairing interactions, and condenses the RNA through interaction with nucleocapsid and integrase to form a mature RNP.
The coarse-grain modeling method used here builds on previous lattice-based methods for modeling bacterial nucleoids [14]. Briefly, the method begins with an initial geometric model of the gRNA built within a sphere representing the Gag polyprotein lattice in the immature virion, and assigns integrase crosslinking sites based on proximity of the experimentally-determined binding sites. The model is then condensed, driven by the weak attraction of nucleocapsid and gRNA, while being constrained by integrase crosslinks and steric bulk of nucleocapsid. Three initial gRNA configurations were tested, based on three different hypotheses for the capture and maturation of the gRNA. “Self-avoiding Gag” models deposit the two gRNA strands on the inner surface of the immature Gag lattice in a self-avoiding random walk, assuming that integrase crosslinking occurs soon after release of the gRNA. This model explores the hypothesis that local features of the RNA placement on the Gag lattice may be retained as the complex condenses. “Overlapping Gag” models similarly deposit the two gRNA strands on the Gag lattice, but allow them to overlap. “Random” models assume that the RNA is released in the immature virion and is randomly distributed within the available volume before condensation. Integrase is known to adopt multiple oligomeric states [15], so separate models with integrase tetramers and with integrase dimers were tested, as well as models without integrase. Coarse-grain models of the mature HIV-1 RNP show a uniform, condensed form (Fig 2). Volumes decrease by roughly a third to a quarter relative to the initial models (Table 1). The three initial configurations (Random, Self-avoiding Gag, and Overlapping Gag) yield similar volumes of the final models. As seen in Fig 2, the RNP globule easily fits within the experimentally-determined structure of the capsid, and matches closely images of the intact capsid from electron microscopy (see, for example, [1]). Models with integrase tetramers showed the greatest volume, followed by globules with integrase dimers, and models with no integrase were smallest. Control experiments varying the number of dimers and tetramers, and comparing the default square-planar model of integrase with a tetrahedral model, show that the difference in size is a direct consequence of the volume occupied by the integrase (Table 1). For example, the default square planar model for the integrase tetramer includes two steric spheres with 9. 2 nm diameter that may overlap, so the total volume of 35 integrase tetramers would be in the range of 14300 to 28500 nm3. In the Self-avoiding Gag model, adding these values to the model with no integrase gives a range of 66600–80800 nm3, which is consistent with the model with integrase tetramer at 70500 nm3. As a control, we also created models using tetrahedral integrase tetramers composed of two dimers, each using the same representation used for the dimer models (6. 5 nm diameter spheres). The tetrahedral tetramer model shows a volume of 61200 nm3, similar to the model with integrase dimers at 61900 nm3. Finally, we did an experiment that would be consistent with rapid exchange of nucleocapsid, by creating the condensed model of RNP without NC, and then choosing IN positions in the condensed globule. The resultant globule (69800 nm3) showed a similar volume as the globule with integrase crosslinking throughout condensation (70500 nm3). Contact probability plots (Fig 3) reveal subtle differences between the three types of models, and these are further quantified by plotting the average value of the contact probability as a function of the separation of nucleotides within or between chains (Fig 4). Models built from the Random configurations show a random sprinkling of interactions between all regions of the gRNA, and a similar distribution of contacts between the two chains. Regions immediately adjacent to the diagonal are sparsely populated, showing the lack of organization at small scales, such as plectonemic supercoils or hairpins. Conversely, models with the Self-avoiding Gag configuration show more interaction near the diagonal. This is expected, since the contact probability of a random chain decays less rapidly with loop length when constrained in 2D compared with 3D [16]. Cross-chain interactions, on the other hand, are reduced due to the self-avoiding definition of the model. Models starting from the Overlapping Gag configurations show a reversed nature: the interaction along the diagonal is slightly reduced when compared to models starting with the Self-avoiding Gag configuration, but the cross-chain interactions are enhanced, presumably due to the many points of close proximity between chains in the initial model. As expected, all three models show a strong interaction at the 5’UTR, due to the modeled secondary structure. A subtle band of reduced interaction extends horizontally and vertically from the diagonal at the 5’UTR, indicating that the 5’UTR forms fewer than expected interactions with the rest of the chains. As shown below, this is a consequence of a general exclusion of the 5’UTR from the body of the globule. We also calculated contact probability plots that are averaged over the two strands, which are more indicative of results that may be obtained from a hypothetical high-resolution contact experiment (Fig 3B). The underrepresented band extending from the 5’UTR is distinguishable, but the more global features that differ between the three models are not as distinguishable between the three plots. Given the coarse-grain nature of the model, the hairpin loops in the 5’UTR do not show a typical double helical structure, but rather end up looking more like distorted bobby pins (see the examples in Fig 5). We noticed early in this study that the secondary structure has a consequence on the global nature of the RNP: during the process of condensation, the 5’UTR is excluded from the body of the globule and often ends up on the surface. We quantified this exclusion with a simple metric, by evaluating the average radial distance of nucleotides in the 5’UTR as compared with the rest of the gRNA and the sites of integrase interaction (Table 2). The central column of this table shows that the average radius of the bulk of the RNA is fairly constant across the default model and several controls, with models with IN tetramers at just over 18 nm, and models with IN dimers or no IN slightly smaller. The right column shows that the IN-binding sites show a similar trend across the default and control models. The 5’UTR shows different behavior, however, based on the assumptions made for the nature of region. Several control experiments help to identify the cause of the exclusion. We tested two hypotheses: the role of NC, and the role of the secondary structure itself. In the default experiments (“FullModel” in Table 2), NC is excluded from the 5’UTR (apart from one site determined experimentally), providing less attractive force for the condensation, resulting in eccentric location of the 5’UTR and larger radial average values. When hypothetical models are condensed with NC covering the 5’UTR as well as the rest of the gRNA (“FullAllNC” in Table 2), the 5’UTR instead is located in the interior of the globule, showing smaller radial average values. This is expected, since the local concentration of nucleocapsid is higher due to the close proximity of the RNA chains when they base pair. When the two RNA chains are treated as separate chains, with no secondary structure, they show a similar behavior with respect to NC. If NC is excluded from the 5’UTR (“TwoChain” in Table 2), the 5’UTR tends to pack on the surface of the globule, and if NC is equally distributed (“TwoAllNC” in Table 2), the 5’UTR has the same properties as the rest of the chain. From these experiments, we conclude that NC is the primary cause of the exclusion of the 5’UTR under the assumptions made by our modelling method.
One of the major goals of this work is to create plausible models of the RNP to identify experimental modalities that could distinguish between different hypotheses for the effect of integrase crosslinking on the final form of the RNP. Note that our protocol is not meant to simulate the process of condensation, rather, it is designed to provide a rapid method for generating multiple models that are consistent with the available data defining the nature of the RNP. The three types of models tested here (Self-avoiding Gag, Overlapping Gag, and Random) are designed to explore different hypotheses for the initial structure of the gRNA and the possibility that IN crosslinking could trap features of this structure in the condensed RNP. Contact probably plots reveal differences in the arrangement of chains in the mature RNP, with the self-avoiding model showing stronger partitioning of the two chains in different regions of the RNP. The study also reveals that, because the two gRNA chains are identical, these differences would be difficult to quantify in a hypothetical Hi-C type experiment (Fig 3B). Two additional morphological features are revealed in these models, which may be accessible to study by cryoEM or super-resolution microscopy. First, crosslinking by integrase leads to a larger condensed globule. Control experiments revealed that this increase in size is primarily due to the steric bulk of the incorporated integrase subunits. However, the model for integrase used here is very simple, with no constraints on the relative orientation of the gRNA strands that are bound. We might expect that the condensed globule may be larger if integrase is more rigid than modeled here, with reduced mobility in the linker to the RNA-binding C-terminal domain and consequently stronger constraints on the orientation of the gRNA binding sites. Unfortunately, there currently are no convincing models of integrase structure at this stage in viral life cycle, but based on available structures of integrase dimers and intasomes (see Methods), we might expect that the connection to the C-terminal domains is quite flexible. Our control experiments suggest that we would not expect a smaller condensed globule if integrase is found to exchange sites rapidly during the process of condensation. The second emergent feature of the models is the exclusion of the 5’UTR from the bulk of the RNP globule. Control experiments implicate the binding propensity of NC for single-stranded nucleic acids [17] as the cause of this exclusion. Exposure of the 5’UTR might be expected to have functional consequences, for example, by allowing ready access to reverse transcriptase for the initiation of genomic DNA synthesis. The current model includes only gRNA, nucleocapsid and integrase, and secondary structure only in the 5’UTR. There are many opportunities for future studies to explore additional functional features and their emergent effects on RNP globule structure and partitioning. These will include a more detailed study of secondary structure, starting first with the Rev response element and extending to more detailed models as defined by SHAPE data. In addition, other molecules, such as transfer RNA and reverse transcriptase, are known to interact with the gRNA and could have effects on the form and function of the RNP. We are also currently developing methods for generating full atomic models from these coarse-grain representations, for use both in simulation and educational outreach.
A lattice-based approximation of the convex hull was used to estimate the volume of RNP globules, and calculated similarly to previous work [14]. The average radial distance of all beads, or a selected subset of beads, relative to the center of gravity was used as a metric to quantify both compactness of a globule and asymmetry in placement of selected portions of the RNA chains. Contact probability plots were calculated by counting the number of contacts within a given threshold (here, 10 nm) within a given sequence bin, across all 25 instances of a particular model. Nine sets of models of the condensed HIV-1 RNP, using the three hypotheses for the starting model, and with integrase tetramers, dimers or no integrase, are available at https: //zenodo. org/record/2662964. Open-source software and input data files for generating models with the self-avoiding Gag hypothesis are available on GitHub at https: //github. com/dsgoodsell/HIVnucleoid.
The genome of HIV-1 is composed of two strands of RNA that are packaged in the mature virion as a condensed ribonucleoprotein complex with nucleocapsid, integrase, and other proteins. We have generated models of the HIV-1 ribonucleoprotein that integrate experimental results from multiple structural and biophysical experiments, exploring several hypotheses about the state of the RNA before condensation, and the role of crosslinking by integrase. The models suggest that the 5’UTR, which shows extensive secondary structure, has a propensity to be placed on the surface of the condensed globule, due to reduced binding of nucleocapsid to double-stranded regions within the 5’UTR. This unexpected localization of the 5’UTR may have consequences for the subsequent structural transitions that occur during the process of reverse transcription.
medicine and health sciences
condensation
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condensed matter physics
microbiology
nucleocapsids
viral structure
retroviruses
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rna structure
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phase transitions
molecular biology
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macromolecular structure analysis
Integrative modeling of the HIV-1 ribonucleoprotein complex