Enhanced HIV infectivity and changes in GP120 conformation associated with viral incorporation of human leucocyte antigen class I molecules

117

135

Accumulating evidence indicates that human immunodeficiency virus type 1 (HIV-1) acquires various cellular membrane proteins in the lipid bilayer of the viral envelope membrane. Although some virionincorporated cellular membrane proteins are known to potently affect HIV-1 infectivity, the virological functions of most virion-incorporated membrane proteins remain unclear. Among these host proteins, we found that CD63 was eliminated from the plasma membranes of HIV-1-producing T cells after activation, followed by a decrease in the amount of virion-incorporated CD63, and in contrast, an increase in the infectivity of the released virions. On the other hand, we found that CD63 at the cell surface was preferentially embedded on the membrane of released virions in an HIV-1 envelope protein (Env)-independent manner and that virionincorporated CD63 had the potential to inhibit HIV-1 Env-mediated infection in a strain-specific manner at the postattachment entry step(s). In addition, these behaviors were commonly observed in other tetraspanin proteins, such as CD9, CD81, CD82, and CD231. However, L6 protein, whose topology is similar to that of tetraspanins but which does not belong to the tetraspanin superfamily, did not have the potential to prevent HIV-1 infection, despite its successful incorporation into the released particles. Taken together, these results suggest that tetraspanin proteins have the unique potential to modulate HIV-1 infectivity through incorporation into released HIV-1 particles, and our findings may provide a clue to undiscovered aspects of HIV-1 entry.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Modulation of Human Immunodeficiency Virus Type 1 Infectivity through Incorporation of Tetraspanin Proteins

Sato

Aoki

Misawa

et al. 2008

117

135

“…This suggests that modification of only a small number of envelope proteins present on the surface of the virus is sufficient to change viral infectivity. It is also possible that deglycosylation of virion-associated producer cell proteins such as adhesins, which are known to play a role in viral infectivity (7,8,12,15,16,25,39), contributed to the differences in infectivity seen in these experiments.…”

Section: Discussionmentioning

confidence: 99%

Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases

Means

Desrosiers

2000

Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations of N-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-␤-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and ␤-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-␤-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.

“…1 (Fig. 1B) and other adherence factors (2,11,15,44) suggest that nearly all virions enter slowly compared to dissociation (40,43). We tested these kinetic interpretations by measuring rates of HIV-1 JRCSF entry by using the CCR5 antagonist TAK779 to inactivate virions that had not completed the CCR5-dependent steps of entry (40,41).…”

mentioning

confidence: 99%

“…A corollary is that titers measured for these viruses may be generally limited by dissociation. Furthermore, dissociation is probably a limiting factor in all cultures because adhesion factors, including ICAM-1 incorporated into HIV-1, greatly increase infectivities for cells, including peripheral blood mononuclear cells (PBMCs) that contain corresponding receptors (2,11,15,44,48), and exogenous factors, including lectins and a semen factor that tightly bind virions onto cells also enhance infectivities (16,26,35,49). Although we conclude that slow diffusion and rapid dissociation severely limit HIV-1 infections in cultures, these kinetic processes are probably less limiting in the context of cell-to-cell transmission and in small intercellular spaces in vivo (7,18,38,46).…”

mentioning

confidence: 99%

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

Platt

Kozak

Durnin

et al. 2010

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