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.
Many viruses, particularly neurotropic alphaherpesviruses such as herpes simplex virus (HSV), require an intact microtubule network for efficient replication and pathogenesis. In living cells, host and viral cargo show rapid reversals in transport direction, suggesting that they can recruit motors of opposing directionality simultaneously. To elucidate the molecular mechanisms for specific motor-cargo recognition, it is necessary to characterize the surface of such cargos. We established a cell-free system that reconstitutes the binding of native, mammalian microtubule motors to intact tegumented HSV capsids. Our data suggest that the inbound motor dynein and the outbound motor kinesin-1 bind directly and independently of other host factors to the inner tegument that coats the capsids during cytosolic transport. Identifying viral receptors for the hosts' transport machinery will provide us on the one hand with new potential targets for antiviral therapy. On the other hand, such viral protein domains could be added to viral vectors or even to artificial nano carriers designed to deliver therapeutic genes or molecules to the nucleus or other subcellular destinations.