Structural analysis of intracellular transport, nuclear entry and egress of Herpes simplex virus 1 by cryo electron tomography

Herpes simplex virus 1 (HSV? 1) is a complex enveloped double stranded DNA virus. The capsid containing the DNA is surrounded by a thick layer of tegument proteins and a lipid envelope with surface glycoproteins. In this work, cryo? electron tomography (cryo? ET), in combination with fluorescence microscopy, was applied to structurally analyze different stages of the HSV? 1 `life? cycle in situ under close? to native conditions and without any chemical fixation. The focus of this study was on intracellular viral transport, attachment of capsids to nuclear pores and viral egress from cells. After entry into host cells by membrane fusion, HSV? 1 capsids are devoid of most of their tegument and preserve the so? called inner tegument. Capsids are then retrogradely transported towards the nucleus along microtubules, most probable by the cellular molecular motor complex dynein? dynactin. After replication, progeny capsids exit the nucleus and are anterogradely transported towards the cell membrane, most likely by the cellular molecular motor kinesin? 1. At the final assembly site of the mature virus (virion) the capsids encounter membranous compartments that are associated with some of the tegument and viral glycoproteins. As a result, virions are further transported towards the cell membrane inside these compartments to exit the cell. Transport of capsids was structurally analyzed in vivo and in vitro by cryo? ET and subtomogram averaging. Some of the inner tegument proteins are assumed to be essential for motor complex binding to the capsid during transport. However, the specific location of the inner tegument proteins and the attachment site of the motor complex have remained in dispute. Here it was revealed that the inner tegument was localized at the capsid vertices, binding to pentons and peripentonal hexons. This suggested that the motor complex binds only at the capsid vertices. Surprisingly, not only capsids containing DNA were transported in vivo, but also capsids devoid of DNA. Significant differences in tegumentation could be observed between these two groups. An in vitro system was established to study the attachment of incoming capsids to nuclear pores. Extracted Xenopus laevis oocyte nuclei that were incubated with isolated capsids were spread and vitrified for cryo? ET observations. Several co? factors that potentially influence the attachment (cytosol, importin? s and ATP) were tested. Secondary envelopment, assembly and egress are the least understood processes in the HSV? 1 `life? cycle. Cryo? ET of infected neurons provided new insights on the molecular structure of these events. An implication of clathrin during secondary envelopment and exit was revealed, as well as a specific orientation of the viral particles against the exit site. The formation of L? particles (noninfective viral particles produced during infection) was also characterized and showed many similarities to the egress pathway in virions. The structural analysis of virus? host cell interactions in vivo and in vitro provided new relevant biological insights into the HSV? 1 `life? cycle, complementing existing biochemical data.