contributed equally to this work Mature, infectious HIV-1 particles contain a characteristic cone-shaped core that encases the viral RNA and replication proteins. The architectures of mature virions and isolated cores were studied using cryoelectron microscopy. The average size (~145 nm) of the virion was unchanged during maturation. Most virions contained a single core but roughly one-third contained two or more cores. Consideration of the capsid protein concentration during core assembly indicated that core formation in vivo is template-mediated rather than concentration-driven. Although most cores were conical, 7% were tubular. These displayed a stacked-disc arrangement with 7-, 8-, 9-or 10-fold axial symmetry. Layer line ®ltration of these images showed that the capsid subunit arrangement is consistent with a 9.6 nm hexamer resembling that previously seen in the helical tubes assembled from puri®ed capsid protein. A common re¯ection (1/3.2 nm) shared between the tubular and conical cores suggested they share a similar organization. The extraordinary¯exi-bility observed in the assembly of the mature core appears to be well suited to accommodating variation and hence there may be no single structure for the infectious virion.

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6 gag , a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6 gag . Consistent with this, viruses with mutations in PR or p6 gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome͞ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.