Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.Viruses must overcome the diffusion barrier of the cytoplasm to effectively replicate in mammalian cells. This is most dramatically exemplified with neurotropic infections, such as those of the alphaherpesviruses, during which virus particles may translocate several centimeters or more between axon terminals and neuronal cell bodies. Intracellular transport of alphaherpesvirus particles to the nucleus in both neurons and non-neuronal cells is dependent on microtubules (20,24,27,40).The alphaherpesvirus virion is composed of four structural elements. The viral genome consists of a linear doublestranded DNA (ca. 120 to 230 kbp) that is housed within a proteinaceous capsid having icosahedral symmetry (ϳ120-nm diameter). The capsid is enclosed within a host-derived lipid envelope, and between the capsid and the envelope is a collection of viral proteins collectively referred to as the tegument. Upon entry into a cell the viral envelope fuses with the cellular plasma or endosomal membrane, depositing the capsid and tegument into the cytosol (10,28,29). At this phase, many tegument proteins are removed from the capsid. However, at least three tegument proteins (VP1/2, UL37, and US3) remain associated with capsids as they travel toward the nucleus (12, 23). After replication and assembly of capsids in the nucleus, progeny capsids translocate to the cytosol where they are again found associated with the VP1/2, UL37, and US3 tegument proteins (8, 13). These capsid/tegument complexes ultimately bud into a component of the secretory pathway and egress from the cell (reviewed in reference 26). The dynamics of capsid transport and assembly in the cytoplasm are poorly understood.Although many alphaherpesvirus proteins can interact with cellular microtubule-based motors, no herpesvirus proteins are currently known to be required for capsid transport (3,4,6,19,25,31,47). The presence of VP1/2, UL37, and US3 on cytosolic capsids makes them prime candidates as effectors of intracellular capsid transport. Of particular interest to the present study, cells infected with viruses lacking either VP1/2 or UL37 assemble genome-containing capsids in the nucleus, and these capsids egress to the cytoplasm similar to capsids of wild-type viruses. However, once in the cytoplasm, unenveloped capsids lacking either VP1/2 or UL37 accumulate, and re-envelopment is rare or nonexistent (1, 2, 9, 16, 17...

How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.All herpesviruses share a common structure that includes a capsid core surrounded by a tegument layer, the latter consisting of viral and cellular proteins derived from the cytosol of the infected cell (10, 40). The capsid and tegument are enveloped by a lipid membrane derived from the cellular secretory pathway (reviewed in reference 39). Although the tegument layer was initially thought to lack defined structure, electron cryomicroscopy and tomography studies of the alphaherpesviruses have determined that the tegument interacts with the capsid surface exclusively at vertices where it adopts the underlying fivefold symmetry (22, 60). The occurrence of many specific interactions between tegument proteins, as well as between tegument proteins and membrane glycoproteins, suggests that the assembly of tegument components directs the production of virions by linking the capsid to emerging envelopes in the infected cell (9,14,16,21,26,27,32,34,42,48,59,62). In accordance with these findings, a direct interaction between specific tegument and capsid proteins of the alphaherpesviruses should exist but has eluded discovery.Here, we show that the carboxy terminus of the VP1/2 tegument protein is a capsid-binding domain (CBD) that is functionally conserved in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). Using a screen to identify viral proteins required for assembly of VP1/2 onto capsids, we identify a minor capsid protein previously found to participate in DNA encapsidation (UL25) as the binding partner for VP1/2 (2, 37). The interaction between the UL25 capsid protein and the VP1/2 tegument protein defines new functions for both of these proteins and reveals a critical missing link in the morphogenesis of the alphaherpesviruses. MATERIALS AND METHODSSubcloning. The green fluorescent protein (GFP) fusion to the carboxy-terminal VP1/2 conserved domain...

The herpesvirus tegument is a layer of viral and cellular proteins located between the capsid and envelope of the virion. The VP1/2 tegument protein is critical for the propagation of all herpesviruses examined. Using an infectious clone of the alphaherpesvirus pseudorabies virus, we have made a collection of truncation and in-frame deletion mutations within the VP1/2 gene (UL36) and examined the resulting viruses for spread between cells. We found that the majority of the VP1/2 protein either was essential for virus propagation or did not tolerate large deletions. A recently described amino-terminal deubiquitinase-encoding domain was dispensable for alphaherpesvirus propagation, but the rate of propagation in an epithelial cell line and the frequency of transport in axons of primary sensory neurons were both reduced. We mapped one essential domain to a conserved sequence at the VP1/2 carboxy terminus and demonstrated that this domain sufficient to redirect the green fluorescent protein to capsid assemblons in nuclei of infected cells.