Replication of human immunodeficiency virus type 1 (HIV-1) in diverse conditions limiting for viral entry into cells frequently leads to adaptive mutations in the V3 loop of the gp120 envelope glycoprotein. This has suggested that the V3 loop limits the efficiencies of HIV-1 infections, possibly by directly affecting gp120-coreceptor affinities. In contrast, V3 loop mutations that enable HIV-1 JR-CSF to use the low-affinity mutant coreceptor CCR5(Y14N) are shown here to have negligible effects on the virus-coreceptor affinity but to dramatically accelerate the irreversible conformational conversion of the envelope gp41 subunits from a three-stranded coil into a six-helix bundle. This slow step is blocked irreversibly by the inhibitor T-20. To further evaluate the role of entry rates in controlling infection efficiencies and viral adaptations, we developed methods to quantitatively measure viral entry kinetics. The virions were adsorbed by spinoculation at 4°C onto HeLa-CD4/CCR5 cell clones that either had limiting or saturating concentrations of CCR5. After warming to 37°C, the completion of entry was monitored over time by the resistance of infections to the competitive CCR5 inhibitor TAK-779. Our results suggest that the efficiency of entry of cell-attached infectious HIV-1 is principally controlled by three kinetic processes. The first is a lag phase that is caused in part by the concentrationdependent reversible association of virus with CD4 and CCR5 to form an equilibrium assemblage of complexes. Second, this assembly step lowers but does not eliminate a large activation energy barrier for a rate-limiting, CCR5-dependent conformational change in gp41 that is sensitive to blockage by T-20. The rate of infection therefore depends on the fraction of infectious virions that are sufficiently saturated with CCR5 to undergo this conformational change and on the magnitude of the activation energy barrier. Although only a small fraction of fully assembled viral complexes overcome this barrier per hour, the ensuing steps of entry are rapidly completed within 5 to 10 min. Thus, this barrier limits the overall flow rate at which the attached virions enter cells, but it has no effect on the lag time that precedes this entry flow. Third, a relatively rapid and kinetically dominant process of viral inactivation, which may partly involve endocytosis, competes with infectious viral entry. Our results suggest that the V3 loop of gp120 has a major effect on the rate-limiting coreceptordependent conformational change in gp41 and that adaptive viral mutations, including V3 loop mutations, function kinetically by accelerating this inherently slow step in the entry pathway.Human immunodeficiency virus type 1 (HIV-1) invasion of cells involves a sequence of steps that ultimately cause fusion of the viral membrane with the cell surface membrane and entry of viral cores into the cytosol (3,4,17,34,48), but the rates of these steps and of the competing viral inactivation processes are unknown. Initially, the virus diffuses onto cell ...

By using immunofluorescence microscopy to observe and analyze freshly made HIV-1 virions adsorbed onto cells, we found that they are inherently highly infectious, rather than predominantly defective as previously suggested. Surprisingly, polycations enhance titers 20-to 30-fold by stabilizing adsorption and preventing a previously undescribed process of rapid dissociation, strongly implying that infectivity assays for many viruses are limited not only by inefficient virus diffusion onto cells but also by a postattachment race between entry and dissociation. This kinetic competition underlies inhibitory effects of CCR5 antagonists and explains why adaptive HIV-1 mutations overcome many cell entry limitations by accelerating entry.It is widely believed that retroviruses, including HIV-1, are predominantly defective, with fewer than 0.1% in plasma or culture media being infectious (4,23,24,30,45). However, other evidence indicates that diffusion severely limits virion contact with cultured cells and that forcing virions onto cells by spinoculation or magnetic methods greatly increases titers (6,17,25,36,45). Additionally, polycations, including DEAEdextran and polybrene, substantially increase titers (5,12,13,20,25,47). Reverse transcription is also somewhat inefficient, depending on the virus isolate and cells used. To reconcile these observations and more accurately measure infectivities, we adsorbed HIV-1 onto highly susceptible JC.53 cells (33, 42) at 4°C by spinoculation or brief incubation with concentrated virus, and we analyzed cell-attached virions by immunofluorescence and deconvolution microscopy (29,39). Figure 1A shows fields of adsorbed HIV-1. Cells were fixed for 10 min with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) containing 2% sucrose and then rinsed and incubated for 10 min in 0.5% NP-40 in PBS containing 10% sucrose, and virions were stained using anti-p24 Gag mouse hybridoma 183 (from the NIH AIDS Research and Reference Reagent Program; provided by B. Chesebro). The Z-stack of deconvoluted images was compressed to show all virions. The fluorescent foci had similar intensities, implying that they were single virions. Accordingly, their staining required extraction of lipids with NP-40 (Fig. 1A, lower panels), and the numbers of foci were directly proportional to virus concentrations in the media (Fig. 1A, inset). Similar results were obtained using HIV-1 labeled with Vpr-green fluorescent protein (GFP), and correlative electron microscopy confirmed that these fluorescent foci were single virions (results not shown).We measured the infectivities of cell-attached HIV-1 following spinoculation (4°C, 30 min, 188 ϫ g) onto cultures pretreated with

SUMMARYBinding of the HIV-1 envelope glycoprotein gp120 to the CCR5 coreceptor reduces constraints on the metastable transmembrane subunit gp41, thereby enabling gp41 refolding, fusion of viral and cellular membranes, and infection. We previously isolated adapted HIV-1 JRCSF variants that more efficiently use mutant CCR5s, including CCR5(Δ18) lacking the important tyrosine sulfatecontaining amino terminus. Effects of mutant CCR5 concentrations on HIV-1 infectivities were highly cooperative, implying that several may be required. However, because wild-type CCR5 efficiently mediates infections at trace concentrations that were difficult to accurately measure, analyses of its cooperativity were not feasible. New HIV-1 JRCSF variants efficiently use CCR5 (HHMH), a chimera containing murine extracellular loop 2. The adapted virus induces large syncytia in cells containing either wild-type or mutant CCR5s and has multiple gp120 mutations that occurred independently in CCR5(Δ18)-adapted virus. Accordingly, these variants interchangeably use CCR5 (HHMH) or CCR5(Δ18). Additional analyses strongly support a novel energetic model for allosteric proteins, implying that the adaptive mutations reduce quaternary constraints holding gp41, thus lowering the activation energy barrier for membrane fusion without affecting bonds to specific CCR5 sites. In accordance with this mechanism, highly adapted HIV-1s require only one associated CCR5 (HHMH), whereas poorly adapted viruses require several. However, because they are allosteric ensembles, complexes with additional coreceptors fuse more rapidly and efficiently than minimal ones. Similarly, wild-type HIV-1 JRCSF is highly adapted to wild-type CCR5 and minimally requires one. The adaptive mutations cause resistances to diverse entry inhibitors and cluster appropriately in the gp120 trimer interface overlying gp41. We conclude that membrane fusion complexes are allosteric machines with an ensemble of compositions, and that HIV-1 adapts to entry limitations by gp120 mutations that reduce its allosteric hold on gp41. These results provide an important foundation for understanding the mechanisms that control membrane fusion and HIV-1's facile adaptability.

Previously it was found that when an acoustic source is located above a porous ground plane of infinite lateral extent, the acoustic fields contain both surface wave and ground wave components. Because of these components the acoustic fields exhibit attenuation differing from the 6 dB per doubling of distance found for propagation over a hard surface. In this paper we consider the excess attenuation that occurs in the mixed path case when a portion of the ground plane is acoustically hard and the rest is porous. Expressions are derived for the attenuation when source and receiver are at ground level and on opposite sides of the boundary line between the hard and porous surfaces. Two different approaches were used to approximate the acoustic fields, and were found to give good numerical agreement. The theoretical results were also found to agree with measurements made at 1 kHz over a concrete–grass path.

The CCR5 coreceptor amino terminus and extracellular (ECL) loops 1 and 2 have been implicated in HIV-1 infections, with species differences in these regions inhibiting zoonoses. Interactions of gp120 with CD4 and CCR5 reduce constraints on metastable envelope subunit gp41, enabling gp41 conformational changes needed for infection. We previously selected HIV-1JRCSF variants that efficiently use CCR5(Δ18) with a deleted amino terminus or CCR5(HHMH) with ECL2 from an NIH/Swiss mouse. Unexpectedly, the adaptive gp120 mutations were nearly identical, suggesting that they function by weakening gp120's grip on gp41 and/or by increasing interactions with ECL1. To analyze this and further wean HIV-1 from human CCR5, we selected variants using CCR5(HMMH) with murine ECL1 and 2 sequences. HIV-1JRCSF mutations adaptive for CCR5(Δ18) and CCR5(HHMH) were generally maladaptive for CCR5(HMMH), whereas the converse was true for CCR5(HMMH) adaptations. The HIV-1JRCSF variant adapted to CCR5(HMMH) also weakly used intact NIH/Swiss mouse CCR5. Our results strongly suggest that HIV-1JRCSF makes functionally critical contacts with human ECL1 and that adaptation to murine ECL1 requires multiple mutations in the crown of gp120's V3 loop.