Rapid Dissociation of HIV-1 from Cultured Cells Severely Limits Infectivity Assays, Causes the Inactivation Ascribed to Entry Inhibitors, and Masks the Inherently High Level of Infectivity of Virions

Abstract

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

Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry

“…Entry was terminated at different times by adding an excess of the CCR5 antagonist TAK779. As reported previously (24), control virions entered in two phases, with a small burst within 1-2 h and the majority entering at a slower sustained rate for ∼20 h ( Fig. 2A).…”

“…Consequently, the previous results could have been caused indirectly by inhibiting these entry steps, thereby enabling more dissociation. To test that possibility, we stabilized adsorption by pretreating cultures with DEAE-dextran before incubating with culture media containing HIV-1 JRCSF for 30 min at 37°C and then counting virions (24). Control virions and virions that were preincubated with 2G12 for 60 min at 37°C bound equally onto cells (Table 1).…”

“…Conversely, adaptive mutations centered in the V3 region of gp120 overcome these limitations and accelerate entry (18,25). Recently, we found that the enhancement of titers caused by pretreating cultures with polycations results from the stabilization of virion adsorption, thus preventing a previously unknown competing process of rapid desorption and providing a longer opportunity for infection (24). These analyses demonstrate that titers in JC.53 cultures are limited by kinetic competition between entry and processes causing virus inactivation.…”

“…These reporters provide a facile means to monitor infections but have no effect on efficiencies of infection by diverse HIV-1 isolates or sensitivities to NAbs (23). This standard assay is done in the presence of DEAE-dextran, a polycation that increases virion adsorption and enhances HIV-1 titers ∼20-fold (20,22,24). We prefer to stain and count infected foci in JC.53 cultures using antibodies to HIV-1, which allows us to compare titers in cell clones having distinct amounts of wild-type or mutant CD4 and/or CCR5 (18,21,25).…”

“…By systematically analyzing this assay system we found that HIV-1 infectivities depend on kinetics of adsorbed virion entry (24,25). Many factors that reduce titers including limiting CD4 and/or coreceptor concentrations or using mutant coreceptors or coreceptor antagonists slow entry (25).…”