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Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The portal channel is a key component in the life cycle of bacteriophages and herpesviruses. The bacteriophage P22 portal is a 1 megadalton dodecameric oligomer of gp1 that plays key roles in capsid assembly, DNA packaging, assembly of the infection machinery, and DNA ejection. The portal is the nucleation site for the assembly of 39 additional subunits generated from multiple copies of four gene products (gp4, gp10, gp9, and gp26), which together form the multifunctional tail machine. These components are organized with a combination of 12-fold (gp1, gp4), 6-fold (gp10, trimers of gp9), and 3-fold (gp26, gp9) symmetry. Here we present the 3-dimensional structures of the P22 assembly-naive portal formed from expressed subunits (gp1) and the intact tail machine purified from infectious virions. The assembly-naive portal structure exhibits a striking structural similarity to the structures of the portal proteins of SPP1 and phi29 derived from X-ray crystallography.

DNA is packaged into preformed bacteriophage capsids to liquid crystalline density by the action of a portal protein complex. Single molecule packaging studies indicate that this is a new and extremely powerful class of molecular motors.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.

A computer model is described for studying the kinetics of the self-assembly of icosahedral viral capsids. Solution of this problem is crucial to an understanding of the viral life cycle, which currently cannot be adequately addressed through laboratory techniques. The abstract simulation model employed to address this is based on the local rules theory of. Proc. Natl. Acad. Sci. USA. 91:7732-7736). It is shown that the principle of local rules, generalized with a model of kinetics and other extensions, can be used to simulate complicated problems in self-assembly. This approach allows for a computationally tractable molecular dynamics-like simulation of coat protein interactions while retaining many relevant features of capsid self-assembly. Three simple simulation experiments are presented to illustrate the use of this model. These show the dependence of growth and malformation rates on the energetics of binding interactions, the tolerance of errors in binding positions, and the concentration of subunits in the examples. These experiments demonstrate a tradeoff within the model between growth rate and fidelity of assembly for the three parameters. A detailed discussion of the computational model is also provided.

The pressure stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure was examined using light scattering, fluorescence emission, polarization, and lifetime methodology. The monomeric protein is very unstable toward pressure and undergoes significant structural changes at pressures as low as 0.5 kbar. These structural changes ultimately lead to denaturation of the subunit. Comparison of the protein denatured by pressure to that in guanidine hydrochloride suggests that pressure results in partial unfolding, perhaps by a domain mechanism. Fluorescence lifetime measurements indicate that at atmospheric pressure the local environments of the tryptophans are remarkably similar, suggesting they may be clustered. In contrast to the monomeric protein subunit, the protein when polymerized into procapsid shells is very stable to applied pressure and does not dissociate with pressure up to 2.5 kbar. However, under applied pressure the procapsid shells are cold-labile, suggesting they are entropically stabilized. The significance of these results in terms of virus assembly are discussed.

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.