SummaryThe actin and microtubule cytoskeleton play important roles in the life cycle of every virus. During attachment, internalization, endocytosis, nuclear targeting, transcription, replication, transport of progeny subviral particles, assembly, exocytosis, or cellto-cell spread, viruses make use of different cellular cues and signals to enlist the cytoskeleton for their mission. Viruses induce rearrangements of cytoskeletal filaments so that they can utilize them as tracks or shove them aside when they represent barriers. Viral particles recruit molecular motors in order to hitchhike rides to different subcellular sites which provide the proper molecular environment for uncoating, replicating and packaging viral genomes. Interactions between subviral components and cytoskeletal tracks also help to orchestrate virus assembly, release and efficient cell-to-cell spread. There is probably not a single virus that does not use cytoskeletal and motor functions in its life cycle. Being well informed intracellular passengers, viruses provide us with unique tools to decipher how a particular cargo recruits one or several motors, how these are activated or tuned down depending on transport needs, and how cargoes switch from actin tracks to microtubules to nuclear pores and back.

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 ...

After viral fusion, capsids of the neurotropic herpes simplex virus are transported along microtubules (MT) to the nuclear pores for viral genome uncoating, nuclear transcription and replication. After assembly and egress from the nucleus, cytosolic capsids are transported to host membranes for secondary envelopment or to the axon terminal for further viral spread. Using GFP-tagged capsids, Cy3-labelled MT and cytosol, we have reconstituted viral capsid transport in vitro. In the presence of ATP, capsids moved along MT up to 30 mm. Blocking the function of dynactin, a cofactor of dynein and kinesin-2, inhibited the transport. Removing outer tegument proteins from the capsids increased in vitro motility. In contrast, capsids isolated from infected nuclei that were devoid of inner as well as outer tegument proteins showed little interaction with dynein and its cofactor dynactin. Our data suggest that the inner tegument of alphaherpesviruses contains viral receptors for MT motors. Herpes simplex virus type 1 (HSV1) is a neurotropic human alphaherpesvirus that initially infects the oral or perioral skin and mucosa before amplified virus enters local sensory and autonomic nerve endings (1). An HSV1 virion consists of a DNA-containing capsid that is covered by about 20 different capsid-associated and tegument proteins, and capsid and tegument are enveloped by a viral membrane (2). Most likely, capsids lose their envelope before moving to the neuronal cell bodies located in cranial ganglia (3-5). The viral dsDNA genome of 152 kb is injected into the nucleoplasm through the nuclear pore (6) and establishes a lifelong latent infection. Upon reactivation, progeny capsids, and possibly virions, contained in membrane vesicles are transported anterogradely to the peripheral nerve endings (7-12). In rare cases, reactivated virus is instead transported to the central nervous system causing life-threatening encephalitis (13).It has been predicted that it would take an HSV1 capsid with a diameter of 125 nm 231 years to diffuse 10 mm in the axonal cytoplasm (14). Instead of diffusion, viral particles use the host cytoskeleton for fast intracellular transport (1,15,16). Microtubules (MT) are long cytoskeletal filaments with biochemically distinct ends assembled from a/b-tubulin (17). The fast-growing plus-ends of MT usually point towards the plasma membrane and in neuronal axons towards the nerve terminals. The lessdynamic MT minus-ends are often stabilized by attachment to a MT-organizing centre located close to the nucleus. HSV1 loses its envelope during cell entry by fusion with the plasma membrane or with an endocytic membrane (18,19), and in epithelial as well as in neuronal cells, incoming capsids, possibly with associated tegument proteins, are transported along MT to the nucleus (20-23). Efficient virus assembly and egress also depend on MT (24,25), and progeny virus uses MT for efficient axonal transport to the synapse (11).Cytoplasmic dynein together with its cofactor dynactin powers most transport to MT minus-ends (26),...

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SummaryAs the inner tegument proteins pUL36 and pUL37 of alphaherpesviruses may contribute to efficient intracellular transport of viral particles, we investigated their role in cytosolic capsid motility during assembly of herpes simplex virus type 1 (HSV1). As reported previously for pUL36, untagged pUL37 and UL37GFP bound to cytosolic capsids before these acquired outer tegument and envelope proteins. Capsids tagged with CheVP26 analysed by live cell imaging were capable of directed long-distance cytoplasmic transport during the assembly of wild-type virions, while capsids of the HSV1-DUL37 or HSV1-DUL36 deletion mutants showed only random, undirected motion. The HSV1-DUL37 phenotype was restored when UL37GFP had been overexpressed prior to infection. Quantitative immunoelectron microscopy revealed that capsids of HSV1-DUL37 still recruited pUL36, whereas pUL37 did not colocalize with capsids of HSV1-DUL36. Nevertheless, the cytosolic capsids of neither mutant could undergo secondary envelopment. Our data suggest that pUL36 and pUL37 are important prior to their functions in linking the inner to the outer tegument. Efficient capsid transport to the organelle of secondary envelopment requires recruitment of pUL37 onto capsids, most likely via its interaction with pUL36, while capsid-associated pUL36 alone is insufficient.

Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-⌬VP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-⌬VP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-⌬VP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.Virions, subviral particles, and viral proteins are actively transported during cell entry, assembly, and egress (17,33,64,65,73,76). Early in infection many viruses use microtubules (MT) for efficient nuclear targeting, either for cytosolic transport of naked viral particles or for transport inside vesicles (16), e.g., herpes simplex virus type 1 (HSV-1) (77), human cytomegalovirus (58), human immunodeficiency virus (48), adenovirus (42, 80), parvoviruses (71, 79), simian virus 40 (61), influenza virus (41), or hepatitis B virus (29).MT are polar cytoskeletal filaments assembled from ␣-/␤-tubulin with a very dynamic plus-end and a less dynamic minusend. N-type kinesins carry cargo towards the MT plus-ends and are involved in transport of viral particles to the plasma membrane during egress (37,39,66,87). Cytoplasmic dynein in cooperation with its cofactor dynactin or C-type kinesins catalyzes transport towards MT minus-ends (60,70,85). In many cell types, the MT minus-ends are clustered at the MT-organizing center (MTOC), which is often in close proximity to the nucleus. Cytoplasmic dynein is required for nuclear targeting of human immunodeficiency virus reverse transcription complexes (48), and capsids of adenovirus (42, 80), canine parvovirus (79), and HSV-1 (18).The HSV-1 virion consists of four structural components: a double-stranded DNA genome of 152 kbp, a capsid shell with a diameter of 125 nm, the tegument, and a membranous envelope (67). The major morphological units of the icosahedral capsid are (i) th...

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-enddirected motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole ( Many viruses, including adenoviruses (Ads) and herpesviruses, spread by intracellular transport within infected host cells, thus increasing the viral load in target organs and possibly causing severe disease (44). The 51 human Ad serotypesclassified into six species (A to F)-have distinct tropisms (19). For example, Ad type 2 (Ad2) and Ad5 (species C) and Ad3 (species B) are associated with upper-airway infections. Other serotypes are linked to epidemic keratoconjunctivitis (species D), pneumonia (species E), enteric infections (species A and F), or infections of hematopoietic cells (41). The Herpesviridae family consists of the alpha-, beta-and gammaherpesviruses. Like Ads, alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), infect different cell types both in cultures and in their hosts. After infection of mucosal or damaged cutaneous epithelium, these neurotropic viruses establish latent infections, primarily in sensory ganglia, that, upon reactivation, lead to recurrent epidermal lesions (40, 55). The ability to infect a broad range of postmitotic cells has made both Ads and herpesviruses useful gene delivery vehicles (21) that are currently being evaluated in clinical trials (29,39). For their application as therapeutic vectors and to identify new potential targets for antiviral therapy, it is crucial to understand how the genomes are targeted to the nucleus.The entry mechanisms for Ads and herpesviruses have been well studied. Ads are internalized by receptor-mediated endocytosis that is dependent on F actin and leave the endosomal pathway at various sites (recently reviewed in reference 11). The species C Ads, including Ad2 and Ad5, exit from a slightly acidic compartment of pH 6 at about 10 min postentry (16, 42), whereas Ad7 (species B) has been reported to escape from acidic late endosomes and lysosomes (32). In contrast, HSV-1 delivers its capsids into the cytosol upon fusion of the viral envelope with the plasma membrane (45,46). Both viruses then target their capsids to the cell nucleus, uncoat, and inject the enclosed linear double-stranded DNA genomes through the nuclear pores into the nucleoplasm for replication (14,35,53). A number of electron microscopy studies have shown that cytoplasmic capsids of both species C Ads and alpha...

To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as ␤-galactosidase expression cassettes. When the BAC pHSV1(17 ؉ )blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17 ؉ )blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17 ؉ )blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.Herpes simplex virus type 1 (HSV1) causes severe human diseases such as herpes encephalitis or herpes keratoconjunctivitis (18). Its double-stranded DNA genome of 152 kb that codes for more than 80 open reading frames is enclosed in an icosahedral capsid with a diameter of 125 nm. HSV1 is a spherical, enveloped virus with a diameter of about 250 nm. Between the capsid and the viral membrane, there is an amorphous, asymmetric layer, the tegument, which consists of about 20 different proteins (45,74). HSV1 enters cells by fusion at the plasma membrane or after endocytosis by fusion with an endosomal membrane (19,42,66,67,82). After dynein-mediated transport along microtubules (32,56,59,81), capsids reach the nuclear pore where the viral genome is released into the nucleoplasm (68) for viral transcription and DNA replication (74). Progeny viral genomes are packaged into preassembled nuclear capsids, which translocate to the inner nuclear membrane. The subsequent steps of herpesvirus morphogenesis are controversial (12, 13, 65).HSV1 capsids can leave the nucleus by primary envelopment at the inner nuclear membrane (6, 64). According to the luminal or single-envelopment hypothesis, these enveloped virions present in the lumen of the nuclear envelope or the endoplasmic reticulu...