Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC ( M orphological maturation represents a key event in the replication cycle of retroviruses. Human immunodeficiency virus type 1 (HIV-1) particles are released as immature, noninfectious virions whose main constituents are the structural polyproteins Gag and Gag-Pro-Pol. Cleavage of Gag and Gag-Pro-Pol at multiple sites by the virus genome-encoded PR (protease) releases the mature structural proteins MA (matrix), CA (capsid), and NC (nucleocapsid) as well as the functional viral enzymes PR, reverse transcriptase (RT), and IN (integrase) (1). Proteolytic processing triggers a dramatic rearrangement of the virion architecture. In immature HIV-1, an incomplete sphere made of ϳ2,500 radially arranged Gag molecules lines the viral lipid envelope. In contrast, mature, infectious particles are characterized by a cone-shaped capsid assembled from CA molecules. The capsid encases a dense ribonucleoprotein (RNP) core, in which NC condenses the viral RNA genome (2, 3).Hexagonal arrangements of the CA domain represent the basic structural element of both the immature Gag lattice and the mature capsid. However, contact surfaces involved in CA-CA interactions differ significantly between immature and mature structures, and the spacing of the immature hexagonal lattice (80 Å) is tighter than that of the mature lattice (96 Å) (2, 3). Furthermore, only about 50% of the CA molecules packaged are used for formation of the mature capsid (4). These observations led to the proposition that morphological maturation occurs via dissociation of the immature lattice, followed by new assembly of the mature cone structure. This model is supported by structural analyses of HIV-1 derivatives carrying mutations at individual PR recognition sites in Gag, in particular, those preventing the late step of CA-SP1 processing (5, 6).Whereas immature HIV-1 particles are very heterogeneous with respect to diameter and the degree of Gag shell completeness (7,8), the majority of mature particles contain a single capsid core structure and a small number of particles contain two capsid structures (9). It therefore appears to be intuitive that the process of capsid assembly from dissociated CA mono-or oligomers

The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly.

Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.