Putative Terminase Subunits of Herpes Simplex Virus 1 Form a Complex in the Cytoplasm and Interact with Portal Protein in the Nucleus

Abstract

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocatesHerpesvirus procapsids and concatameric viral DNA accumulate in the nuclei of infected cells. The procapsids consist of a roughly spherical proteinaceous shell surrounding an inner protein shell or scaffold (16,24,36). To initiate DNA packaging, an enzyme called the terminase is believed to scan the viral DNA in search of genomic ends, cleave the concatemer into single genomes, engage the procapsid at a portal vertex designed for the passage of the DNA, and drive the genome into capsids through the hydrolysis of ATP.Current evidence supports the hypotheses that the herpes simplex virus (HSV) terminase comprises the products of U L 15, U L 28, and U L 33 (pU L 15, pU L 28, and pU L 33, respectively), whereas the portal vertex consists of a dodecamer of the U L 6 protein (pU L 6). These hypotheses are supported by the observations that (i) pU L 6, pU L 15, pU L 28, and pU L 33 are each essential for DNA packaging (2,5,25,26,34,44); (ii) epitopes of these proteins are present on the external surface of viral capsids, and at least pU L 15 and pU L 28 are associated with procapsids (23, 31, 41); (iii) pU L 15 interacts with the pU L 28 moiety of a pU L 28/pU L 33 complex in infected cells (9,18,19,43); (iv) pU L 15 contains an ATPase-like motif that is essential for viral replication (13, 45); (v) pU L 28 binds DNA sequences known to be required for the formation of normal DNA termini (1, 17); and (iv) pU L 6 forms a dodecameric ring in vitro with a size and conformation that match the dimensions of capsid vertices and portal vertices of some bacteriophages (23, 37).The main focus of the current study concerns a key question that distinguishes two models of DNA packaging: specifically, whether the terminase engages the portal vertex in the cytoplasm or in the nucleus. If the terminase were to engage the portal in the cytoplasm, it follows that portal assembly into the procapsid would also incorporate the bound terminase. This would imply that the entire procapsid, with incorporated terminase, would then scan viral DNA in search of genomic ends. On the other hand, if the terminase were imported into the nucleus separately from the portal, it would be free to scan the DNA independently of the procapsid and, once bound to target DNA sequences, could engage the portal vertex for eventual DNA cleavage and translocation into the capsid. The latter mechanism is similar to that used by many bacteriophage terminases (4,6,11).Where the HSV terminase forms in the cell and where the portal and terminase interact have been addressed previously using transient expression assays. For example, transiently expressed pseudorabies virus pU L 28 localizes in the cytoplasm unless coexpressed with HSV type 1 (HSV-1) pU L 15, suggesting that pU L 15 is responsible for the import of the terminase complex (20). On the other hand, pU L 6 was also shown to import pU L 28 into the nucleus when the proteins were coexpressed, and mutations that ...

Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits

“…Together these three factors form a complex that was purified from HSV-1-infected cells (26) and identified using immunoblot analysis in cells infected with HCMV (88). Growing evidence supports a model where the terminase complex assembles in the cytoplasm, and its translocation to the nucleus is dependent on L-terminase NLS (61,83). Our bioinformatic analysis of pUL15 NLS suggests this scenario is certainly possible for ␣-herpesviruses (Fig.…”

“…functional NLS was previously identified and validated at the N terminus of HSV-1 L-terminase subunit (pUL15) (61) and at the C terminus of HCMV S-terminase (pUL56) (62) (Fig. 1A).…”

DNA Virus Replication Compartments

“…Typically, this process involves proteins forming an internal scaffold where the capsid shell is assembled and the genome is subsequently incorporated (99)(100)(101). Thereafter, the assembled nucleocapsid exits the nucleus by a unique budding mechanism through the nuclear membrane.…”

The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

“…To date, the knowledge about these HSV-1 proteins is limited, and no function has been assigned to their HCMV counterparts. HSV-1 pUL33 interacts with the HSV-1 terminase proteins pUL15 and pUL28 (2) as well as with the portal vertex pUL6, suggesting that pUL33 may help in stabilizing the interaction between the terminase subunits and/or in translocating viral DNA into the capsid (1,41). HSV-1 pUL25 is not required for DNA cleavage but is required for efficient encapsidation (23,34).…”