Function of Dynein and Dynactin in Herpes Simplex Virus Capsid Transport

Abstract

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus. INTRODUCTIONTo initiate a successful infection, animal viruses bind to the cell surface, penetrate into the cytosol, and target their genome to the sites of viral transcription and replication. For many viruses this is the host nucleus (Whittaker et al., 2000). Particular neurotropic viruses that enter at the presynaptic plasma membrane, such as herpes simplex viruses, are transported over long distances because the site of entry is far away from the nucleus. Herpes simplex virus type 1 (HSV1) is a human pathogen that initially replicates in epithelial cells of the oral cavity. Amplified virus enters neurons and is transported to the neuronal nuclei located in the trigeminal ganglion (reviewed in Enquist et al., 1998). After lytic infection of some neurons, a latent infection is established (Wagner and Bloom, 1997).We have calculated that it would take 231 years for a herpes virus capsid to diffuse by 10 mm in the axonal cytoplasm (Sodeik, 2000). High concentrations of protein, the cytoskeleton, and organelles cause molecular crowding in the cytoplasm, which effectively restricts free diffusion of molecules larger than 500 kDa (Luby-Phelps, 2000). Thus, virions and subviral particles are transported by active processes. Besides hijacking vesicular transport during endocytosis and secretion, viruses also exploit the host's cytoskeleton directly for their itinerary (Sodeik, 2000;Ploubidou and Way, 2001).HSV1 virions consist of four structural components: DNA, capsid, tegument, and envelope (Steven and Spear, 1997;Zhou et al., 2000). The icosahedral capsid with a diameter of 125 nm surrounds the double-stranded viral DNA of 152 kb. The tegument, the hallmark of all herpes viruses, is an amorphous layer of ϳ20 proteins. It is localized between the capsid and the viral envelope that contains ϳ12 membrane ...

Virus trafficking – learning from single-virus tracking

“…2). This was observed for Reovirus, Adenovirus, HSV, influenza virus and HIV 5,[42][43][44][45] . Viral transport is often highly regulated as with, for example, HSV in neurons.…”

“…Viral transport is often highly regulated as with, for example, HSV in neurons. After fusion of incoming virions with the plasma membrane, HSV capsids are transported towards the minus end of microtubules by dynein and its cofactor dynactin 43 . In the axons of sensory neurons, incoming HSV capsids are moved by retrograde transport, whereas newly assembled capsids undergo bidirectional and salutatory motions, indicating a modulation of the plus-end directed motility 46 .…”

Transport and egress of herpes simplex virus in neurons

“…This transport requires intact MTs, and can be disrupted by colchicine, vinblastine or nocodazole, in both non-polarised cells [49] and sensory neurons [50]. Incoming HSV-1 capsids associate with cytoplasmic dynein and dynactin, and their transport to the nucleus is dynein dependent, since it can be blocked by the over-expression of the dynactin subunit p50 (dynamitin) [51].…”

“…The relative contribution of other tegument or capsid proteins to HSV-1 transport is not known, although it has been suggested that the major tegument protein pUL36, or one of the minor capsid proteins such as pUL25, may also contribute to dynein binding in vivo [51,67]. In the case of PrV, the so-called inner tegument proteins pUL36, pUL37 and pUS3 have been shown to remain associated with the capsid during entry HSV transport and egress HSV transport and egress 39 39 [68,69].…”

A quantitative measure for alterations in the actin cytoskeleton investigated with automated high‐throughput microscopy

“…Before seeding, cells were washed and detached using 3 mM EDTA in PBS. A total of 3 3 10 4 cells was seeded into a well of a 8-chambered Lab-Tek cover glass (Nunc), coated with bovine fibronectin (Sigma Aldrich), and allowed to settle down for 3 h. Subsequently, they were incubated for another 40 min with cytochalasin D or latrunculin B (Biomol) prior to fixation and permeabilization according to the PHEMO fixation protocol (33). Cells were blocked for 30 min with 1% BSA in PBS and stained for actin and microtubules using a ORIGINAL ARTICLE monoclonal mouse-anti-a-tubulin antibody (Cell Signal), a rabbit-anti-mouse IgG coupled to Atto-633 secondary antibody (Sigma Aldrich), and phalloidin-TRITC (Invitrogen) at a concentration of 1 lg/ml.…”