SummaryAs the inner tegument proteins pUL36 and pUL37 of alphaherpesviruses may contribute to efficient intracellular transport of viral particles, we investigated their role in cytosolic capsid motility during assembly of herpes simplex virus type 1 (HSV1). As reported previously for pUL36, untagged pUL37 and UL37GFP bound to cytosolic capsids before these acquired outer tegument and envelope proteins. Capsids tagged with CheVP26 analysed by live cell imaging were capable of directed long-distance cytoplasmic transport during the assembly of wild-type virions, while capsids of the HSV1-DUL37 or HSV1-DUL36 deletion mutants showed only random, undirected motion. The HSV1-DUL37 phenotype was restored when UL37GFP had been overexpressed prior to infection. Quantitative immunoelectron microscopy revealed that capsids of HSV1-DUL37 still recruited pUL36, whereas pUL37 did not colocalize with capsids of HSV1-DUL36. Nevertheless, the cytosolic capsids of neither mutant could undergo secondary envelopment. Our data suggest that pUL36 and pUL37 are important prior to their functions in linking the inner to the outer tegument. Efficient capsid transport to the organelle of secondary envelopment requires recruitment of pUL37 onto capsids, most likely via its interaction with pUL36, while capsid-associated pUL36 alone is insufficient.

The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating. Infections with herpes simplex virus type 1 (HSV1; human alphaherpesvirus 1) cause the common herpes labialis, herpes keratitis, and keratoconjunctivitis, as well as life-threatening neonatal infections, herpes encephalitis in patients with primary immune deficiencies, and eczema herpeticum in patients with atopic dermatitis (46,54,101,102). The virions contain the DNA genomes of 152 kb encased in icosahedral capsids that interact with the surrounding tegument; this protein layer consists of a partially icosahedrally ordered inner portion and a less organized outer portion that connects to the viral lipid envelope (42,88,101,118). HSV1 packages up to 26 different tegument proteins that have been grouped into inner and outer tegument on the basis of their preferred association with capsids or membranes during assembly and entry as well as their fractionation behavior during virion lysis (40,60,62,68,75,96,116).Herpesvirus morphogenesis commences in the nucleus, where preassembled capsids package newly synthesized viral genomes (12,33,47,75). According to the most widely accepted secondary reenvelopment model, nuclear capsids traverse the nuclear membranes by primary envelopment at the inner nuclear membrane and primary fusion with the membranes of the endoplasmic reticulum to enter the cytosol. Inner tegument proteins may bind to nuc...

dCleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment.

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ⌬UL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ⌬UL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ⌬UL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ⌬UL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.The infection cycle of human cytomegalovirus (HCMV) comprises a phase in the cell nucleus where genome replication and assembly of new capsids take place (24). Replication of the 230-kbp viral DNA genome leads to the formation of concatemers of head-to-tail-linked viral genomes, which are believed to be highly branched. These concatemers are subsequently cleaved into unit-length genomes, which are packaged into preformed capsids. The DNA-filled capsids associate with some tegument proteins at the nuclear membrane and then are transferred into the cytoplasm, where they undergo further coating with tegument proteins. Final envelopment of the capsids most likely occurs in a cytoplasmic virus assembly compartment, which partly overlaps with and is possibly derived from the trans-Golgi network (30).Cleavage-packaging of HCMV genomes and capsid maturation in the nucleus are not completely understood, yet several viral proteins have been implicated as involved in these processes. The HCMV terminase responsible for cleavage of concatemeric DNA was shown to consist of two essential proteins, pUL56 and pUL89 (31, 36). pUL56 binds to viral capsids as well as to the packaging signal located in the a-repeat of the HCMV genome and has been shown to possess ATPase activity. pUL89 directly interacts with pUL56 and seems to be required mainly for DNA cleavage (4,19,31). pUL104 i...