This was accomplished through tabulation of the percentage of image pixels that were greater than two standard deviations brighter than the mean brightness value for each image. or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of assembly also can dismantle pre-existing tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature HIV-1 core assembly, and avenues for antiviral inhibition. Keywords: HIV, retrovirus, capsid, Gag, assembly Introduction The mechanisms by which HIV-1 capsid (CA) proteins assemble to form mature-type cores and are not known. Within virions, CA proteins assemble mostly conical but occasionally cylindrical cores using a CA N-terminal domain (NTD) hexamer organization that is similar to the one observed in sheets, spheres, and cylinders (tubes) assembled assembly of mature-type cores from purified HIV-1 CA proteins is a simpler, but still poorly understood process. Wild type (WT) HIV-1 CA proteins, which dimerize via their C-terminal domains (CTDs) with a Kd of about 18 uM22, can be induced by salt treatment to assemble long tubes, as well as rare cone and sphere Bedaquiline (TMC-207) forms3,6-7,10-12,16,18,20. Experiments have demonstrated that a W184A mutation at the CTD dimer interface inhibits dimerization, and blocks salt-induced CA assembly16. In contrast, it has been shown that deletion of CA residues 87-97 (87-97), within the NTD cyclophilin A (CypA) binding loop, dramatically Bedaquiline (TMC-207) increases the efficiency of tube assembly16,23-24. assembly reactions have been enlisted to examine the effects of potential HIV assembly inhibitors25-32. One such inhibitor, the peptide CAI (ITFEDLLDYYGP28-32), was shown to inhibit HIV-1 Gag and CA assembly reactions when present in a 5-fold molar excess relative to the viral proteins, but has been presumed to be ineffective against pre-assembled cores28-29. Interestingly, CAI binds to a CTD site that ordinarily interacts with NTD helix 4 residues, facilitating the alignment of NTDs and CTDs around hexamer rings20,33. Although analysis of inhibitors and ROCK2 mutations has contributed to an improved understanding of the CA protein contacts required for core assembly3-4,6-8,10-11,16,25-32,34-40, much of the assembly pathway remains to be elucidated. A speculative model is depicted in Figure 1. As illustrated, the model shows a nucleation step (Figure 1, step 1 1), followed by a growth phase (steps 2 and 3). Because EM images of assembly incubations often show either no assembly products or a preponderance of reasonably long Bedaquiline (TMC-207) tube products2-3,6,8,10-13,16,18, we assume that the nucleation step is Bedaquiline (TMC-207) slow, relative to a rapid, energetically favored growth phase. However, it is important to emphasize that nucleation limited kinetics have not been demonstrated rigorously for the pathway, and that many other details are also uncertain. For instance, the subunits that form the nucleation complex and that are added during the growth phase could be CA monomers, dimers, or higher order oligomers. The composition of the nucleation complex also is unknown. Additional unresolved issues are whether tube growth is unidirectional or bidirectional, whether growing ends can be capped, and how tube lengths and widths are controlled. Open in a separate window Figure 1 HIV-1 core assembly modelShown is a model for the assembly from HIV-1 capsid proteins, in which nucleation limited kinetics are assumed, although this has not been proven rigorously. As illustrated in step 1 1, protein subunits oligomerize to form a nucleation complex (gray cluster of subunits) that serves to promote the rapid growth of tubes, depicted in steps 2 and 3. Note that although the model shows assembly in one direction, bidirectional assembly is not excluded. Note also that the nature of the subunits that combine to form the nucleation complex, the composition of the nucleation complex, and the sizes of the subunits that are added during the rapid growth phase are unknown. One of the impediments to elucidation of the CA assembly pathway is the cumbersome nature of the Bedaquiline (TMC-207) available assays. For accurate qualitative characterization of assembly incubations, EM analysis frequently is employed2-19, although quantitation of results from the relatively small areas monitored by EM is problematic. In contrast, the efficiencies of CA assembly reactions can be followed via pelleting or turbidity assays25-26,28-29,39,41, but these methods are subject to the constraint that they can.
