The product of the herpes simplex virus type 1 UL28 gene is essential for cleavage of concatemeric viral DNA into genomelength units and packaging of this DNA into viral procapsids. To address the role of UL28 in this process, purified UL28 protein was assayed for the ability to recognize conserved herpesvirus DNA packaging sequences. We report that DNA fragments containing the pac1 DNA packaging motif can be induced by heat treatment to adopt novel DNA conformations that migrate faster than the corresponding duplex in nondenaturing gels. Surprisingly, these novel DNA structures are high-affinity substrates for UL28 protein binding, whereas double-stranded DNA of identical sequence composition is not recognized by UL28 protein. We demonstrate that only one strand of the pac1 motif is responsible for the formation of novel DNA structures that are bound tightly and specifically by UL28 protein. To determine the relevance of the observed UL28 protein-pac1 interaction to the cleavage and packaging process, we have analyzed the binding affinity of UL28 protein for pac1 mutants previously shown to be deficient in cleavage and packaging in vivo. Each of the pac1 mutants exhibited a decrease in DNA binding by U L28 protein that correlated directly with the reported reduction in cleavage and packaging efficiency, thereby supporting a role for the UL28 protein-pac1 interaction in vivo. These data therefore suggest that the formation of novel DNA structures by the pac1 motif confers added specificity on recognition of DNA packaging sequences by the U L28-encoded component of the herpesvirus cleavage and packaging machinery.T he herpes simples virus type 1 (HSV-1) genome is composed of two segments of unique sequence, designated as long (U L ) and short (U S ), that are flanked by inverted repeats (denoted ba and ca, respectively), such that the genome structure can be represented as a n b-U L -bЈaЈ m cЈ-U S -ca, where n and m vary from 1 to more than 10 (1). Generation of linear unit-length genomes from concatemeric viral DNA occurs through specific DNA cleavage within the terminally repeated a sequences (as shown in Fig. 2 A), which contain all of the cis-acting signals required for genome maturation (2-6). The a sequence is composed of a copy of the 20-bp direct repeat (DR1) at each end and two segments of unique sequence U c (58 bp) and U b (64 bp) that are separated by a series of directly repeated (DR) elements. The composition of the internal direct repeats varies between HSV-1 strains but always includes an array of 12-bp DR2 sequences.The specific signals for DNA cleavage and packaging, termed pac1 and pac2, are located within the U b and U c regions, respectively. The pac motifs, which were identified by their conservation 30-35 bp from the termini of mature herpesvirus genomes, have been implicated directly in mediating the cleavage and packaging reaction (7-10). pac1 is characterized primarily by two stretches of five to eight G residues separated by a 3-to 7-nt T-rich region, whereas pac2 contains a conserved C...

In nuclei of cells infected with herpes simplex virus (HSV), synthesized viral DNA accumulates as concatamers that are cleaved into genomic lengths and inserted into preformed capsids. Whereas newly replicated DNA and enzymes required for DNA synthesis accumulate in sites of infected cell nuclei termed replication compartments, the intranuclear site of DNA cleavage and packaging is currently controversial. DNA packaging requires the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes in addition to the major capsid proteins. Using confocal immunofluorescence microscopy, it was observed that in > 95% of HEp-2 cells fixed at late times after infection with wild-type HSV-1, capsids, major capsid proteins ICP5 and ICP35, and the UL6-encoded minor capsid protein localized in DNA replication compartments. These data support the hypothesis that capsid assembly and DNA cleavage/packaging normally occur in HEp-2 cell replication compartments. In contrast, cells infected with a viral mutant lacking functional UL17 contained antigenically dense nuclear aggregates that stained with ICP35, ICP5, and capsid specific antibodies. Cells infected with the UL17 mutant virus also displayed UL6-specific fluorescence in a diffuse pattern at the nuclear periphery in regions not containing ICP35 and ICP5. Displacement of ICP35 from replication compartments was not observed in cells infected with cleavage/packaging mutants lacking UL28 and UL33. We conclude that the UL17 gene is required for correct targeting of capsids and major and minor capsid proteins to the DNA replication compartment of HEp-2 cells and deduce that this targeting reflects one functional role of UL17 in viral DNA cleavage and packaging.

Previous studies have suggested that the UL17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZexpression cassette in place of 1,490 bp of the 2,109-bp UL17 open reading frame [HSV-1(ΔUL17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the UL17 open reading frame [HSV-1(UL17-stop)] were plaque purified on engineered cell lines containing the UL17 gene. A virus derived from HSV-1(UL17-stop) but containing a restored UL17 gene was also constructed and was designated HSV-1(UL17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔUL17) nor HSV-1(UL17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔUL17) or HSV-1(UL17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔUL17) or HSV-1(UL17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔUL17) compared to wild-type virus show no detectable differences. These data indicate that the UL17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the UL17 gene product, an anti-UL17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent M r 77,000 and weakly with a protein of apparent M r 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted UL17 protein. We therefore conclude that the UL17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.

The UL15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against UL15-encoded protein recognized three proteins with apparent M rs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-M rprotein was detected in type C capsids and comigrated with the product of a UL15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-M r proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔUL15), a virus lacking an intact UL15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of UL15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent M rs of 83,000, 80,000, and 79,000 are products of UL15 with identical C termini. The 79,000-, 80,000-, and 83,000-M r proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that UL15-encoded proteins are integral components of B capsids. Only the 83,000-M r protein was detected in B capsids purified from cells infected with viruses lacking the UL6, UL17, or UL28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-M rproteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-M r UL15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.

The UL15 gene of herpes simplex virus type 1 (HSV-1), like UL6, UL17, UL28, UL32, and UL33, is required for cleavage of concatameric DNA into genomic lengths and for packaging of cleaved genomes into preformed capsids. A previous study indicated that the UL15 gene encodes minor capsid proteins. In the present study, we have shown that the amino-terminal 509 amino acids of the UL15-encoded protein are sufficient to confer capsid association inasmuch as a carboxyl-terminally truncated form of the UL15-encoded protein with an M r of approximately 55,000 readily associated with capsids. This and previous studies have shown that, whereas three UL15-encoded proteins with apparent M rs of 83,000, 80,000, and 79,000 associated with wild-type B capsids, only the full-length 83,000-M r protein associated with B capsids purified from cells infected with viruses lacking functional UL6, UL17, UL28, UL32, and UL33 genes (B. Salmon and J. D. Baines, J. Virol. 72:3045–3050, 1998). Thus, all viral mutants that fail to cleave viral DNA into genomic-length molecules also fail to produce capsid-associated UL15 80,000- and 79,000-M r proteins. In contrast, the 80,000- and 79,000-M r proteins were readily detected in capsids purified from cells infected with a UL25 null virus that cleaves, but does not package, DNA. The conclusion that the amino terminus of the 83,000-M r protein is truncated to produce the 80,000- and/or 79,000-M r protein was supported by the following observations. (i) Whereas the C termini of the 83,000-, 80,000-, and 79,000-M r proteins are identical, immunoreactivity dependent on the first 35 amino acids of the UL15 83,000-M r protein was absent from the 80,000- and 79,000-M r proteins. (ii) The 79,000- and 80,000-M r proteins were detected in capsids from cells infected with HSV-1(UL15M36V), an engineered virus encoding valine rather than methionine at codon 36. Thus, initiation at codon 36 is unlikely to account for production of the 80,000- and/or 79,000-M r protein. Taken together, these data strongly suggest that capsid-associated UL15-encoded protein is proteolytically cleaved near the N terminus and indicate that this modification is tightly linked to maturation of genomic DNA.