Transport of human immunodeficiency virus type 1 pseudoviruses across the blood-brain barrier: role of envelope proteins and adsorptive endocytosis

Abstract

Blood-borne human immunodeficiency virus type 1 (HIV-1) crosses the blood-brain barrier (BBB) to induce brain dysfunction. How HIV-1 crosses the BBB is unclear. Most work has focused on the ability of infected immune cells to cross the BBB, with less attention devoted to the study of free virus. Since the HIV-1 coat glycoprotein gp120 can cross the BBB, we postulated that gp120 might be key in determining whether free virus can cross the BBB. We used radioactive virions which do (Env+) or do not (Env-) bear the envelope proteins to characterize the ability of HIV-1 to be taken up by the murine BBB. In vivo and in vitro studies showed that the envelope proteins are key to the uptake of free virus and that uptake was enhanced by wheat germ agglutinin, strongly suggesting that the envelope proteins induce viral adsorptive endocytosis and transcytosis in brain endothelia. Capillary depletion showed that Env+ virus completely crossed the vascular BBB to enter the parenchyma of the brain. Virus also entered the cerebrospinal fluid, suggesting passage across the choroid plexus as well. About 0.22% of the intravenously injected dose was taken up per g of brain. In vitro studies showed that postinternalization membrane cohesion (membrane binding not reversed with acid wash or cell lysis) was a regulated event. Intact virus was recovered from the brain endothelial cytosol and was effluxed from the endothelial cells. These results show that free HIV-1 can cross the BBB by an event related to adsorptive endocytosis and mediated by the envelope proteins.

FIG. 1

Transport of Env⁺ virus, Env⁻ virus, and albumin across the BBB. The transport of Env⁺ virus was faster than that of either albumin or Env⁻ virus. There was no statistically significant difference in the rate of transport of Env⁻ versus that of albumin.

FIG. 2

Clearance of virus from blood after i.v. injection. (A) Clearance of Env⁺ virus. WGA had little effect on clearance. (B) Clearance of Env⁻ virus. WGA had little effect on clearance. Env⁺ (no WGA) is shown for reference.

FIG. 3

Percentage of an i.v. dose of Env⁺ virus taken up per gram of brain. Results have been corrected for the vascular space of the brain. Peak value was about 0.22%/g.

FIG. 4

Effect of WGA on virus uptake. (A) Pilot study showing that WGA had an effect on Env⁺ virus uptake but not on Env⁻ virus uptake; (B) effect of WGA on the uptake of Env⁺ and Env⁻ virus 5 min after i.v. injection. WGA produced a statistically significant enhancement of Env⁺ uptake but not of Env⁻ uptake.

FIG. 5

Viral entry into CSF compartment after i.v. injection of Env⁺. (A) Comparison of brain/serum and CSF/serum ratios; (B) ratios corrected for albumin space. The dashed line represents unity and shows that the albumin-corrected brain/CSF ratio is greater than 1.

FIG. 6

Percentage of total binding by IBM. (A) Comparison of capillary binding of Env⁺ and Env⁻ virus (No WGA); (B) comparison of uptake of Env⁺ virus incubated with or without WGA.

FIG. 7

Fate of Env⁺ virus taken up by IBM. For details of measurements, see Materials and Methods.

FIG. 8

Effect of WGA on fate of Env⁺ taken up by IBM. WGA increased percentages of total binding, reversible binding, and internalization in a dose-response manner. The percentage of membrane coherence decreased with increasing WGA concentration. ∗, P < 0.05 in comparison to no dose.

FIG. 9

Effects of WGA, PS, and PS plus WGA on fate of Env⁺ virus taken up by IBM. WGA, PS, and PS plus WGA increased percentage of total binding and internalization; the percentage of reversible binding was increased only with PS plus WGA. WGA and PS plus WGA but not PS alone decreased the percentage of membrane coherence. ∗, P < 0.05 for indicated comparisons.

FIG. 10

Identification of radioactivity recovered from IBM. Radioactivity was characterized on a 4B-200 Sepharose column. The elution peaks for virus, gp120, and albumin as determined for this type of column are shown. (A) Characterization of radioactivity recovered from the cytoplasm of IBM that had been incubated with Env⁺ virus. Over 50% eluted in the position of virus. (B) Characterization of radioactivity effluxed by IBM that had been preincubated with Env⁺ virus. About 25% of effluxed material eluted in the position of virus, showing that intact virus survives endocytosis and exocytosis to either the luminal or abluminal surface.