HIV-1 infects macrophages by exploiting an endocytic route dependent on dynamin, Rac1 and Pak1.

Recent studies provide compelling evidence that HIV-1 entry in cell lines and lymphocytes proceeds by endocytosis, but these studies are still lacking in macrophages, an important natural target cell for HIV-1. Macrophages exhibit continual and extensive endocytic activity as part of their natural functions, so we investigated the uptake pathways involved in productive HIV-1 entry. We find that caveolae are not utilised by HIV-1, because the main structural proteins, caveolin-1 and 2 are absent from most human leukocytes. We then focused on macropinocytosis; we find that HIV-1 entry into macrophages is sensitive to inhibitors of Na(+)/H(+) exchange, actin rearrangement, dynamin, Rho family GTPases, and Pak1, but not to inhibitors of PI-3 kinase and myosin II. This leads us to conclude that HIV entry into macrophages proceeds by an endocytic pathway that is not classical macropinocytosis. Because of the limitations of a purely pharmacological study such as this, the final elucidation of this pathway awaits the development of reliable forward genetic approaches in authentic macrophages.

HIV entry in macrophages is dependent on intact lipid rafts.

Macrophages are an important natural target cell for HIV-1, but previous studies of virus entry into these cells are limited, and the involvement of membrane cholesterol and lipid rafts is unknown. Cholesterol disruption of macrophage membranes using four pharmacological agents acting by different mechanisms: methyl-beta cyclodextrin, nystatin, filipin complex and Lovastatin, all significantly inhibited productive HIV entry and reverse transcription. The inhibitory effects of these drugs resulted in decreased virus release from infected cells, and could be substantially reversed by the addition of water-soluble cholesterol. The virus bound equally to cholesterol-disrupted cells even though HIV receptor expression levels were significantly reduced. Macrophage CD4 and CCR5 were found to partition with the detergent-resistant membranes with a typical raft-associating protein flotillin-1. HIV particles were observed co-localising with a marker of lipid rafts (CTB-FITC) early post infection. These data suggest that macrophage membrane cholesterol is essential for HIV entry, and implicate lipid raft involvement.

Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane.

The extracellular enveloped virus (EEV) form of vaccinia virus (VACV) is surrounded by two lipid envelopes. This presents a topological problem for virus entry into cells, because a classical fusion event would only release a virion surrounded by a single envelope into the cell. Recently, we described a mechanism in which the EEV outer membrane is disrupted following interaction with glycosaminoglycans (GAGs) on the cell surface and thus allowing fusion of the inner membrane with the plasma membrane and penetration of a naked core into the cytosol. Here we show that both the B5 and A34 viral glycoproteins are required for this process. A34 is required to recruit B5 into the EEV membrane and B5 acts as a molecular switch to control EEV membrane rupture upon exposure to GAGs. Analysis of VACV strains expressing mutated B5 proteins demonstrated that the acidic stalk region between the transmembrane anchor sequence and the fourth short consensus repeat of B5 are critical for GAG-induced membrane rupture. Furthermore, the interaction between B5 and A34 can be disrupted by the addition of polyanions (GAGs) and polycations, but only the former induce membrane rupture. Based on these data we propose a revised model for EEV entry.

Ligand-induced and nonfusogenic dissolution of a viral membrane.

Hitherto, all enveloped viruses were thought to shed their lipid membrane during entry into cells by membrane fusion. The extracellular form of Vaccinia virus has two lipid envelopes surrounding the virus core, and consequently a single fusion event will not deliver a naked core into the cell. Here we report a previously underscribed mechanism in which the outer viral membrane is disrupted by a ligand-induced nonfusogenic reaction, followed by the fusion of the inner viral membrane with the plasma membrane and penetration of the virus core into the cytoplasm. The dissolution of the outer envelope depends on interactions with cellular polyanionic molecules and requires the virus glycoproteins A34 and B5. This discovery represents a remarkable example of how viruses manipulate biological membranes, solves the topological problem of how a double-enveloped virus enters cells, reveals a new effect of polyanions on viruses, and provides a therapeutic approach for treatment of poxvirus infections, such as smallpox.

Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans.

Vaccinia virus (VACV) produces two distinct enveloped virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), but the entry mechanism of neither virion is understood. Here, the binding and entry of IMV particles have been investigated. The cell receptors for IMV are unknown, but it was proposed that IMV can bind to glycosaminoglycans (GAGs) on the cell surface and three IMV surface proteins have been implicated in this. In this study, the effect of soluble GAGs on IMV infectivity was reinvestigated and it was demonstrated that GAGs affected IMV infectivity partially in some cells, but not at all in others. Therefore, binding of IMV to GAGs is cell type-specific and not essential for IMV entry. By using electron microscopy, it is demonstrated that IMV from strains Western Reserve and modified virus Ankara enter cells by fusion with the plasma membrane. After an IMV particle bound to the cell, the IMV membrane fused with the plasma membrane and released the virus core into the cytoplasm. IMV surface antigen became incorporated into the plasma membrane and was not left outside the cell, as claimed in previous studies. Continuity between the IMV membrane and the plasma membrane was confirmed by tilt-series analysis to orientate membranes perpendicularly to the beam of the electron microscope. This analysis shows unequivocally that IMV is surrounded by a single lipid membrane and enters by fusion at the cell surface.

Vaccinia virus cores are transported on microtubules.

Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK(2) cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51.8 (+/-3.9) microm min(-1), consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.