Identifying a New Mechanism of HIV Core Formation
Schematic comparison between the researchers’ newly proposed model (left) and an earlier model for HIV-1 core formation (right). In immature virions, Gag is arranged beneath the viral membrane. In our model, the capsid (CA) layer (yellow) is released by proteolytic cleavage from the matrix (green) and rolls while transforming to the mature lattice, until the mature core is fully formed. In earlier models, proteolytic cleavage is proposed to release CA to the viral lumen, leading to core nucleation and growth in a diffusion-controlled process.
During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the HIV-1 core, and NC packages the viral RNA within the core. Current models of the development of HIV-1 suggest that when CA is cleaved from Gag it dissociates from the membrane and moves into the virus interior before nucleating, in a concentration-dependent manner, into the core, which is the last step in virus maturation. The core is thought to grow from its narrow end stopping only when it reaches the opposite side of the virus membrane. Since blocking the formation of infectious viral particles is an important therapeutic strategy, it is critical to understand the detailed mechanism of core maturation.
Sriram Subramaniam, Ph.D., a Senior Investigator, and Gabriel Frank, Ph.D., a Postdoctoral Fellow in the Laboratory of Cell Biology, and their colleagues used cryo-electron tomography to analyze the three-dimensional structure of viruses and other membrane-bound particles in the supernatants of HIV-1 infected cells. Intriguingly, they observed membrane-enclosed particles with the viral Env protein on their surfaces that contained multiple cores in their interiors. Some of the cores were free-floating, while others were attached to the membrane. The researchers also found immature Gag proteins in the membrane-bound structures suggesting cores can form within these structures. The presence of cores not in contact with the membrane also suggests that core formation does not require membrane at both ends and implies that the dimensions of the virus envelope do not determine core size, counter to the currently proposed nucleation and growth mechanism of core formation.
To verify that the multicore particles did not arise from the fusion of mature virions, the investigators performed the same study with Env negative HIV-1, which renders the virus noninfectious. Again, they saw the same membrane-bound structures with multiple cores. The scientists also found areas of immature Gag and incomplete cores, some with sheet-like structures, that were still attached to the membrane, suggesting that the multicore structures do not form from the fusion of mature particles and that the multicore structures form prior to core maturation.
The researchers next imaged intact cell pellets using transmission electron microscopy (TEM) to confirm that the multicore structures are not an artifact of the purification process. Encouragingly, they still observed particles with more than one core. However, TEM is a two-dimensional method, which hampers the analysis of core size and location. The investigators then examined the original infected cultures with focused ion beam scanning electron microscopy, a three-dimensional method. They again found multicore structures and showed that these structures had a greater proportion of membrane area per core, which would not be expected if their formation were due to virus fusion. The presence of cores with the same general size and shape of those in mature virions also suggests that the dimensions of the membrane-bound structure do not dictate core length, again challenging the current core formation model.
The scientists then visualized the MA lattice on the inner membrane of the multicore structures. They found that the MA layer is arranged hexagonally but was often disordered with gaps in the packing. The MA lattice could be distinguished from the immature CA lattice by their distinct distances from the membrane and the larger size of the MA lattice The MA lattice parameters were similar to those reported for purified MA on artificial membranes, while the parameters for the immature CA lattice corresponded with studies of immature HIV-1 and other lentiviruses. The researchers found that the MA lattice correlated with flatter areas of the membrane and suggest that the multicore structures, which have a larger surface area and less membrane curvature, allow for the presence of the observed extended MA lattice. Using subvolume averaging, the researchers enhanced the detail of the packing arrangement of the MA layer and found that the lattice has hexagonal voids of approximately 6nm. These voids are larger than those observed in the artificial membranes and suggest that in their native confirmation MA trimers interact with adjacent trimers near or at their tips.
The scientists hypothesized that the various core structures they observed were snapshots across the maturation continuum. Examining a number of the transitional stages, they observed a structure in which a sheet of membrane-associated CA was contiguous with a detached and partially rolled mature core-like structure. Another structure showed almost complete cores associated with the membrane along one side rather than at an end. A third revealed immature Gag associated with mature CA lattice pulling away from the membrane. If these are truly intermediate stages of maturation, none could be explained by the growth and nucleation model of core formation. These results led the researchers to propose a new model of core maturation in which CA detaches from the membrane after cleavage from Gag as a mostly intact layer that rolls as it transitions into the mature core lattice structure. This phase transition does not depend on diffusion and would be favored kinetically compared to the growth and nucleation model. Future studies may point to novel therapeutic targets that can inhibit HIV-1 infection based on this unique core maturation mechanism.Summary Posted: 01/2015
Frank GA, Narayan K, Bess JW, Del Prete GQ, Wu X, Moran A, Hartnell LM, Earl LA, Lifson JD, Subramaniam S.Maturation of the HIV-1 core by a non-diffusional phase transition. Nature Communications. January 8, 2015 PubMed Link