Characterization of Herpes Simplex Virus-Containing Organelles by Subcellular Fractionation: Role for Organelle Acidification in Assembly of Infectious Particles

Abstract

The cytoplasmic compartments occupied by exocytosing herpes simplex virus (HSV) are poorly defined. It is unclear which organelles contain the majority of trafficking virions and which are occupied by virions on a productive rather than defective assembly pathway. These problems are compounded by the fact that HSVinfected cells produce virus continuously over many hours. All stages in viral assembly and export therefore coexist, making it impossible to determine the sequence of events and their kinetics. To address these problems, we have established assays to monitor the presence of capsids and enveloped virions in cell extracts and prepared HSV-containing organelles from normally infected cells and from cells undergoing a single synchronized wave of viral egress. We find that, in both cases, HSV particles exit the nucleus and accumulate in organelles which cofractionate with the trans-Golgi network (TGN) and endosomes. In addition to carrying enveloped infectious virions in their lumen, HSV-bearing organelles also displayed nonenveloped capsids attached to their cytoplasmic surface. Neutralization of organellar pH by chloroquine or bafilomycin A resulted in the accumulation of noninfectious enveloped particles. We conclude that the organelles of the TGN/endocytic network play a key role in the assembly and trafficking of infectious HSV.The mechanisms of envelopment and intracellular trafficking of herpes simplex virus (HSV) are poorly understood. A number of ultrastructural studies have shown that viral nucleocapsids containing packaged DNA are assembled in the infected cell nucleus and then bud through the inner nuclear membrane into the perinuclear space, acquiring an envelope (62,63,66). However, the interpretation of images representing subsequent stages in virus egress has been controversial. Electron microscopy reveals that the infected cell cytoplasm contains capsids partially wrapped by adjacent membranes, enveloped virions completely enclosed within membranous vesicles, and "naked" capsids entirely lacking envelopes (4,14,38,41,72).Two principal HSV egress models have been proposed to account for these various cytoplasmic structures (27,80). In one model, it has been suggested that perinuclear enveloped virions enter transport vesicles budding from the outer nuclear membrane or endoplasmic reticulum (ER) and then traffic as normal secretory pathway cargo through the Golgi apparatus and are eventually secreted from the cell (25,38,66,72). In this model, naked cytoplasmic capsids arise due to nonproductive, erroneous deenvelopment, wherein the envelope of trafficking virions becomes fused with the surrounding lipid bilayer of the transport vesicle. Increased accumulation of naked capsids in the cytoplasm of cells infected by an HSV strain mutant in glycoprotein gD supports the view that premature fusion can generate nonenveloped capsids (14); however, it is not clear if such a phenomenon is the source of naked capsids during a wild-type viral infection.In an alternative model (65), perinuclear virions u...

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-inducible cofactor

“…2 shows that after AlF treatment, a portion of hGBP-1 floated to low-buoyant-density fractions 1 and 2, consistent with membrane association. The transGolgi marker TGN 46 and the endosomal marker Rab 5 were also found in these low-density fractions, as anticipated (27). In contrast, I B␣, a cytosolic protein, remained in the lower-gradient fractions.…”

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“…Cells were then trypsinized in the presence of AlF for 5 min at 37°C, washed twice with homogenization buffer (HB, 250 mM sucrose͞2 mM MgCl 2 ͞10 mM Tris, pH 7.6), resuspended in 1.5 ml of HB buffer containing protease inhibitors, and disrupted by using a ballbearing homogenizer (0.0011-inch clearance). Residual cells and debris were pelleted at 1,500 ϫ g for 10 min at 4°C, and 0.9 ml of the postnuclear supernatant was layered onto a 12-ml sucrose gradient (27). After centrifugation in an SW41 rotor at 39,000 rpm (261,000 ϫ g) for 4 h at 4°C, 1-ml fractions were isolated, and trichloroacetic acid-precipitated proteins were separated by 10.5% SDS͞PAGE followed by Western blotting with R.GBP-1, as described (28).…”

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Herpes simplex virus types 1 and 2 modulate autophagy in SIRC corneal cells

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