Herpes simplex virus 1 fuses with the plasma membrane of a host cell, and the incoming capsids are efficiently and rapidly transported across the cytosol to the nuclear pore complexes, where the viral DNA genomes are released into the nucleoplasm. Using biochemical assays, immunofluorescence, and immunoelectron microscopy in the presence and absence of microtubule depolymerizing agents, it was shown that the cytosolic capsid transport in Vero cells was mediated by microtubules. Antibody labeling revealed the attachment of dynein, a minus end–directed, microtubule-dependent motor, to the viral capsids. We propose that the incoming capsids bind to microtubules and use dynein to propel them from the cell periphery to the nucleus.

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.

Abstract. Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cell associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) α and β. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.