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.

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Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particle's center. We related image features to the polyprotein domain structure by combining mutagenesis with cryoelectron microscopy and image analysis. The matrix (MA) domain appears as a thin layer tightly associated with the inner face of the viral membrane, separated from the capsid (CA) layer by a low-density region corresponding to its C terminus. Deletion of the entire p6 domain has no effect on the width or spacing of the density layers, suggesting that p6 is not ordered in immature human immunodeficiency virus type 1 (HIV-1). In vitro assembly of a recombinant Gag polyprotein containing only capsid (CA) and nucleocapsid (NC) domains results in the formation of nonenveloped spherical particles which display two layers with density matching that of the CA-NC portion of immature HIV-1 Gag particles. Authentic, immature HIV-1 displays additional surface features and an increased density between the lipid bilayers which reflect the presence of gp41. The other internal features match those of virus-like particles.

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Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa. The genomes of spumaretroviruses (or foamy viruses [FV])include the classical gag, pro-pol, and env genes that are the hallmark of the retrovirus family (9). Despite the familial relationship implied by their genome organization, FV differ from retroviruses such as oncoviruses or lentiviruses in basic aspects of replication and gene expression (30-32, 39, 53). The differences between FV and the more widely studied retroviruses, such as human immunodeficiency virus (HIV) or murine leukemia virus, provide an opportunity to identify the fundamental mechanisms of processes such as particle assembly and maturation.The shared gene order of the FV genomes and those of other retroviruses allows the identification of common structural proteins. Unfortunately, this relationship does not extend to the structure and function of FV Gag proteins, since no obvious homologies are detectable between FV Gag and MA, CA, and NC domains of other retroviruses for which structures have been determined to high resolution (10, 43). Furthermore, the proteolytic processing of Gag that produces the familiar mature proteins in other retroviruses is unusual, incomplete, and/or delayed in FV (12,23,29,37).The available data indicate that the morphology and morphogenesis of FV are distinct from those of other retroviruses. FV Gag proteins preassemble to form in the cytoplasm spherical capsids that bud through cellular membranes (55). This process contrasts with what occurs with lentiviruses and C-type retroviruses that assemble their capsids at the site of budding but is similar to what occurs with B-and D-type retroviruses (40). Budding of human foamy virus (HFV) i...

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The cellular protein cyclophilin A (CypA) binds specifically to the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and is incorporated into HIV-1 particles at a molar ratio of 1:10 (CypA/CA). Structural analysis of a CA-CypA complex suggested that CypA may destabilize interactions in the viral capsid and thus promote uncoating. We analyzed the influence of CypA on the in vitro assembly properties of wild-type (WT) CA and derivatives containing substitutions of Gly89 in the Cyp-binding loop. All variant proteins were significantly impaired in CypA binding. In the presence of CypA at a molar ratio of 1:10 (CypA/CA), WT CA assembled into hollow cylinders that were similar to those observed in the absence of CypA but slightly longer. Higher CypA concentrations inhibited cylinder formation. Variant CA proteins G89L and G89F yielded similar cylinders as the WT protein but were significantly more resistant to CypA. Cryoelectron microscopic analysis of WT cylinders assembled in the presence of CypA revealed direct binding of CypA to the outer surface. Electron diffraction patterns generated from these cylinders indicated that CypA causes local disorder. The addition of CypA to preassembled cylinders had little effect, however, and cylinders were only disrupted when incubated with a threefold molar excess of CypA for several hours. These results suggest that CypA does not efficiently destabilize CA interactions at the molar ratio observed in the virion and therefore is unlikely to serve as an uncoating factor.