Materials and Methods Strains, cultivation and purification of virions. African green monkey kidney (Vero) cells (CCL-81; ATCC, Mannasas, VA) and human foreskin fibroblasts (HFT) (S1) cells were grown in alpha-minimum essential medium supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA) and passaged as described in Desai et al. (S2). Virus stocks were prepared in Vero cells as described (S2). HFT cells were infected with KOS virus at an MOI of 10 PFU/cell. 48

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated K⌬UL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with K⌬UL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of K⌬UL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In K⌬UL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in K⌬UL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.The herpes simplex virus type 1 (HSV-1) virion is comprised of four structural elements: a DNA-containing core; an icosahedral capsid, which encloses the genome; a layer that immediately surrounds the capsid termed the tegument; and an outer membrane or envelope, which encloses the whole structure and in which are embedded the viral glycoproteins (39, 55; reviewed in references 40 and 47). The tegument represents the most diverse structural element of the virus particle in terms of both polypeptide composition and functions.Virus-specified polypeptides that comprise the tegument structure include those that function to activate transcription, shut off host protein synthesis, and uncoat the virus genome, as well as others whose functions are not yet known (reviewed in references 40 and 47). The role of tegument is twofold. First, the tegument can be envisioned as a structure that delivers factors into the cytosol of the infected cell to facilitate the initiation of a successful infection. Components of the tegument that mediate this process...

Summary Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α-, β-, and γ-herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase critical for virus replication and spread. The extent of substrate conservation and the key common cell signalling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective anti-viral strategies.

The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated K⌬UL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus K⌬UL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, K⌬UL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in K⌬UL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of K⌬UL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of K⌬UL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.The tegument layer of the herpes simplex vir...

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.

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SUMMARYA temperature-sensitive mutant of herpes simplex virus type 1, tsQ26, was shown to contain an amino acid substitution in glycoprotein H (gH). The mutant entered cells efficiently at the non-permissive temperature and replicated to give nearly normal yields of intracellular infectivity. The intracellular virions contained, predominantly, an immature form ofgH and no gH was found on the surface of infected cells. Excreted virions were devoid of gH and were not infectious. Virions excreted at the permissive temperature were infectious and contained gH and no loss of gH resulted from incubation of these virions at the non-permissive temperature. The temperaturesensitive phenotype apparently results from the loss of gH from virions during their transport to the cell surface, and since loss of gH is accompanied by loss of infectivity we conclude that gH is an essential component of the infectious virion.

Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here we demonstrate that cells respond to HSV-1 infection by promoting filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum-dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.