Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.

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),...