Journal of General Virology

Interaction of the herpes simplex virus type 1 packaging protein UL15 with full-length and deleted forms of the UL28 protein

Adrian P. Abbotts, Valerie G. Preston, Michelle Hughes, Arvind H. Patel and Nigel D. Stow

MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK

The UL15 and UL28 proteins of herpes simplex virus type 1 are both required for the packaging of replicated viral DNA into the viral capsid. We have expressed UL28 and a functional epitope-tagged form of UL15 in mammalian and insect cells. Immunoprecipitation experiments confirmed that the two proteins can interact. In agreement with previous results, UL15, when expressed alone, entered the nucleus but UL28 remained cytoplasmic. When co-expressed the two proteins co-localized in the nucleus. Six UL28 deletion mutants were constructed and similarly analysed. The results obtained by immunoprecipitation and immunofluorescence were consistent and demonstrate that at least two separate regions of the UL28 polypeptide chain have the ability to interact with UL15. Surprisingly, three of the mutants prevented the UL15 protein from localizing to the cell nucleus, and these were not functional in a transient DNA packaging assay. Of the three UL28 mutant proteins that entered the nucleus with UL15, one containing an internal deletion of 13 amino acids was able to complement a UL28 null mutant in both DNA packaging and virus yield assays, demonstrating that this region of the protein is not essential for function. In addition to interacting with the UL28 protein we also demonstrated that UL15 molecules can interact with each other, and that sequences within the second exon contribute to this interaction.

The genomes of herpesviruses are linear double-stranded DNAs of 125–245 kbp which replicate in the nuclei of infected cells generating tandem head-to-tail concatemers. During the assembly of progeny particles, the excision of unit length molecules from these concatemers is tightly coupled to their packaging into preformed capsids. A single cis-acting DNA sequence element and at least six proteins, which are well conserved throughout the virus family, appear to play direct roles in the cleavage/packaging process (for a review see Homa & Brown, 1997). In herpes simplex virus type 1 (HSV-1), an alphaherpesvirus, the packaging proteins are encoded by genes UL6, UL15, UL17, UL28, UL32 and UL33. Mutants with lesions in these genes are defective in both cleavage of concatemeric DNA and DNA encapsidation (Homa & Brown, 1997; Salmon et al., 1998; Lamberti & Weller; 1998). A seventh gene product, encoded by UL25, is not required for cleavage but is apparently necessary for retention of DNA by the capsid (McNab et al., 1998), as well as functioning at an early stage during virus entry (Addison et al., 1984).

The functions of the individual proteins in HSV-1 DNA packaging remain poorly understood, but it is anticipated that their roles will be similar to those of analogous proteins of double-stranded DNA bacteriophage (for reviews see Catalano et al., 1995; Fujisawa & Morita, 1997; Catalano, 2000). The latter proteins recognize the DNA to be packaged, assemble a 'packaging complex' at a specific DNA entry site on a preformed capsid, cleave the DNA at an appropriate site to initiate encapsidation, inject it into the capsid and cleave again to terminate the process. The packaging proteins of bacteriophage may be structural components of the particle (e.g. portal proteins at the capsid vertex used for DNA entry), transiently associated during encapsidation (e.g. the terminase enzyme) or may perform catalytic roles in forming the various complexes. A pivotal role is played by the terminase, generally comprised of two proteins, which interacts with both the DNA substrate and the portal vertex, functions as an ATP-driven pump to translocate the genome into the capsid shell and carries out the cleavage reactions.

The precursor capsid for HSV-1 DNA packaging is termed a procapsid and consists of an icosahedral assembly of capsid proteins around a proteinaceous scaffold. B capsids of similar composition and lacking viral DNA accumulate in HSV-1-infected cells. Packaging of DNA into the procapsid results in the release of the scaffold and the formation of a C capsid, which subsequently acquires tegument and envelope to generate the virion (for reviews see Rixon, 1993; Homa & Brown, 1997). To date no portal vertex has been identified but the UL6 and UL25 proteins have been shown to be associated with B capsids, C capsids and virions (Patel & Maclean, 1995; Ali et al., 1996; McNab et al., 1998). In contrast the UL15 and UL28 proteins are found predominantly in B capsids, although a modified form of UL15 may also be present in C capsids and virions (Yu & Weller, 1998 a; Salmon & Baines, 1998; Taus & Baines, 1998). Neither the UL32 nor the UL33 protein was detected in any of the capsid forms (Lamberti & Weller, 1998; Reynolds et al., 2000). UL17 has been described as a virion protein located in the tegument layer surrounding the capsid, but is also found in B and C capsids (Salmon et al., 1998; Goshima et al., 2000).

The involvement of the UL15 and UL28 proteins in HSV-1 DNA packaging was first demonstrated with temperature-sensitive mutant viruses and confirmed through the characterization of null mutants (Addison et al., 1990; Poon & Roizman, 1993; Cavalcoli et al., 1993; Tengelsen et al., 1993; Yu et al., 1997; Baines et al., 1997). Subsequently, several lines of evidence have suggested a direct interaction between the two proteins, possibly analogous to that between the subunits of bacteriophage terminase. Immunofluorescence studies indicated that when expressed alone UL15 exhibited a nuclear localization. In contrast, UL28, or the homologous protein of another alphaherpesvirus, pseudorabies virus (PRV), remained cytoplasmic. Co-expression of HSV-1 UL15 with the HSV-1 or PRV UL28 protein, however, facilitated entry of the latter into the nucleus (Koslowski et al., 1997, 1999). UL15 and UL28 were additionally demonstrated to co-purify from HSV-1-infected cells, apparently as a heterodimer (Koslowski et al., 1999).

Further support is provided by studies utilizing a betaherpesvirus, human cytomegalovirus (HCMV). Mutants resistant to benzimidazole compounds that selectively inhibit processing and packaging of HCMV DNA have been isolated and the increased resistance was shown to result from alterations within the HCMV UL89 and UL56 proteins, the homologues of HSV-1 UL15 and UL28, respectively (Underwood et al., 1998; Krosky et al., 1998). Moreover, HCMV UL56 has been reported to bind to the viral DNA packaging signal, a property in common with the small subunits of several terminases (Bogner et al., 1998). However, this observation awaits confirmation and no similar activity has yet been attributed to HSV-1 UL28. Finally, HSV-1 UL15 and its herpesvirus homologues show limited sequence similarity to gp17, the large subunit of the terminase complex of bacteriophage T4 (Davison, 1992). The similarity includes a consensus ATP-binding site which has been demonstrated by site-directed mutagenesis to be essential for UL15 function (Yu & Weller, 1998 b).

Only one report has attempted to identify regions of UL15 or UL28 important for interaction. These studies demonstrated that the C-terminal 155 amino acids of the PRV UL28 protein were necessary for its nuclear localization in cells superinfected with a PRV UL28 null mutant or co-transfected with HSV-1 UL15 (Koslowski et al., 1997). It is not known, however, whether the complex formed between the heterologous proteins is functional in DNA packaging. In this manuscript we extend these findings by using immunoprecipitation assays to confirm the interaction between the HSV-1 UL15 and UL28 proteins, and examining the ability of a series of UL28 deletion mutants to support DNA packaging and to interact with UL15.

Cells and viruses. Baby hamster kidney 21 clone 13 (BHK) cells were grown in Glasgow MEM supplemented with 10 % tryptose phosphate broth, 10 % newborn calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Vero cells were grown in Dulbecco's MEM containing 5 % foetal calf serum and the same antibiotics. Spodoptera frugiperda (Sf) cells (strain IPLB-SF-21; Kitts et al., 1990) were maintained in TC100 medium supplemented with 5 % foetal calf serum and the same antibiotics. The HSV-1 UL28 null mutant gCB was propagated on the Vero cell-derived complementing cell line, C1 (Tengelsen et al., 1993), and S648, a UL15 null mutant, was propagated in clone 17 cells (Baines et al., 1997). The parental virus used for the construction of recombinant baculoviruses was AcPAK6 (Bishop, 1992). DNA fragments for expression (see below) were cloned into the transfer vector pAcCL29-1 (Livingstone & Jones, 1989) and recombinants were isolated through recombination with Bsu36I-cleaved AcPAK6 DNA essentially as described by Kitts et al. (1990).

Expression of UL28 proteins. Plasmid pUL28 contains the HSV-1 DNA fragment spanning nucleotides 58182 (EagI site; positions from McGeoch et al., 1988) to 55761 (SgrAI site) inserted into the SmaI site of the expression vector pCMV10 (Stow et al., 1993) such that full-length UL28 protein is expressed under the control of the HCMV major immediate early (IE) promoter. A panel of six UL28 gene deletions was made by utilizing convenient restriction endonuclease sites within pUL28 (Table 1). The full-length UL28 fragment was cloned into the SmaI site of the transfer vector pAcCL29-1 in the correct orientation downstream of the polyhedrin gene promoter and the corresponding 2, 3, 4, 5 and 6 deletions were introduced. Recombinant baculoviruses were constructed using the resulting plasmids.

Table 1. UL28 mutants used in these studies

* ds indicates a site in the poly-linker downstream of the UL28 ORF; us indicates a site upstream of the UL28 ORF.

† In the 4 and 5 constructs an XbaI linker (New England Biolabs) with termination codons in all three frames was inserted at the site of the deletion and specifies the C-terminal amino acids LV. In the 6 constructs sequences from the N terminus of the HSV-1 UL8 gene derived from plasmid pE8 (Stow et al., 1993) and specifying a 14 bp upstream untranslated region and the first five amino acids (MDTAD) were inserted at the site of the deletion to provide an in-frame initiation codon.

Expression of UL15 proteins. To express full-length UL15 protein, fragments specifying the first and second exons were first assembled with a synthetic oligonucleotide so as to produce a single fragment equivalent to a cDNA copy of the spliced mRNA (Dolan et al., 1991; A. Davison, unpublished data). This fragment (nucleotides 29010–30048 linked to 33635–35094) was cloned using BglII linkers into a derivative of pCMV10 (pCMV10-BglII) in which the SmaI site had been converted to a BglII site. A plasmid containing the insert in the correct orientation was designated pJM9. Plasmid pJM19, encoding the full-length UL15 protein linked to a C-terminal epitope tag derived from the HCMV UL83 gene product (pp65) was constructed by inserting a synthetic oligonucleotide duplex between the MluI site at position 34801 and the XbaI site in the poly-linker of pJM9. The oligonucleotide was designed so that an epitope tag sequence (ERKTPRVTGG) was added to the authentic C terminus of UL15. To generate plasmid pMH20, which encodes a tagged version of UL15 exon II, a fragment encoding the epitope tag and most of exon II was first excised from JM19. This fragment was inserted into pCMV10-BglII together with an oligonucleotide which specified the remaining sequence and an in-frame ATG initiation codon. The above UL15 gene fragments were also transferred to the baculovirus transfer vector pAcCL29-1 and used to construct recombinant viruses. AcUL15, AcUL15-pp65 and AcUL15E2-pp65 contain the inserts from pJM9, pJM19 and pMH20, respectively.

Antibodies. Purified mouse monoclonal antibody reactive with the HCMV pp65 epitope tag (anti-pp65) was purchased from Capricorn Products (AntiCMV late nuclear protein). Rabbit polyclonal antibody R123 was obtained following immunization with a bacterially expressed protein representing amino acids 138–785 of UL28. Mouse monoclonal antibody 13924, reactive with the HSV-1 UL9 protein, was described previously (Stow et al., 1998).

Transient complementation yield assay. Monolayers of BHK cells in 35 mm Petri dishes (2x10⁶ cells per plate) were transfected with pCMV10-derived expression plasmids by the calcium phosphate procedure followed by treatment with DMSO at 4 h (Stow & Wilkie, 1976). Each monolayer received 1 µg of the indicated plasmid and 12 µg calf thymus carrier DNA. The transfected cells were infected with 5 p.f.u. per cell of the appropriate HSV-1 null mutant in a volume of 200 µl. One hour after virus addition the inoculum was removed and the infectivity of residual virus was inactivated with an acid–glycine wash (Rosenthal et al., 1984). The plates were washed once with 0.14 M NaCl, exposed to 0.1 M glycine, 0.14 M NaCl pH 3.0 for 1 min, washed once with Eagle's medium containing 5 % newborn calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin (EC5), and incubation was continued for 18 h at 37 °C in 2 ml EC5. The cells were scraped into the growth medium, sonicated and the yield of virus was titrated at 37 °C on both Vero cells and the appropriate complementing cell line.

Transient complementation packaging assay. Plasmid pSA1 was constructed by inserting a 200 bp fragment spanning the junction between two tandem 'a' sequences between the HindIII and EcoRI sites of pS1 (Stow & McMonagle, 1983) which contains a copy of the HSV-1 ori_S DNA replication origin. In the presence of wild-type HSV-1 the pSA1 amplicon is both efficiently replicated and packaged into virus particles, confirming that the inserted fragment contains a functional packaging signal (Nasseri & Mocarski, 1988; P. D. Hodge, unpublished data). To examine whether pCMV10-derived expression plasmids could complement the packaging defects of S648 or gCB, monolayers of BHK cells in 35 mm dishes were transfected with 1.0 µg expression plasmid, 0.5 µg pSA1 and 12 µg calf thymus DNA, and infected with null mutant virus as described above, except that the acid–glycine wash was omitted. At 20 h post-infection (p.i.) the cells from each monolayer were resuspended in TBS and divided into two equal samples which were used to prepare total and DNase-resistant (i.e. encapsidated) DNA as described previously (Stow et al., 1983; Stow, 1998). DNA samples were cleaved with EcoRI and DpnI, fractionated by agarose gel electrophoresis, transferred to a Hybond-N membrane (Amersham) and replicated (DpnI-resistant) pSA1 DNA was detected by hybridization to a probe prepared from the plasmid vector pAT153. Phosphorimages of the Southern blots were acquired using the Personal Molecular Imager and Quantity One software (Bio-Rad).

Immunoprecipitation assays. Immunoprecipitation assays were performed as described by McLean et al. (1994). Monolayers of Sf cells (1.2x10⁶ cells per 22 mm diameter tissue culture well) were infected with 5 p.f.u. per cell recombinant baculoviruses and labelled with [³⁵S]L -methionine from 24 to 40 h p.i. Soluble extracts (150 µl per well) were prepared and 130 µl was incubated with 1 µl undiluted R123 or anti-pp65 antibody as indicated. The immune complexes were collected on protein A–Sepharose beads, washed and the proteins separated by SDS–PAGE. To detect labelled proteins, gels were either dried and subjected to phosphorimage analysis or fixed, treated with En3Hance (Du Pont) and exposed to autoradiographic film. Western blots were performed as described by Towbin et al. (1979). The membranes were blocked at room temperature for 90 min using 5 % dried milk in TBS and incubated with anti-UL28 rabbit serum R123 at a dilution of 1/200 in TBS containing 0.1 % Tween-20 and 5 % dried milk (TBSTM). After 90 min, the membrane was washed extensively with TBSTM, incubated for 30 min with alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega, 1/7500 in TBSTM), washed again and bound antibody was detected using a BCIP/NBT liquid substrate system (Sigma).

Immunofluorescence assays. Vero cells were seeded onto glass coverslips in Linbro wells (1.5x10⁵ cells per 13 mm diameter coverslip) 1 day prior to lipofection. Each well received the indicated plasmids (total of 1 µg DNA) and 6 µl lipofectamine (Life Sciences) in 200 µl unsupplemented Dulbecco's MEM. At 16 h post-transfection the cells were fixed with 5 % formaldehyde in PBS containing 2 % sucrose, and permeabilized with 0.5 % NP-40 in PBS with 10 % sucrose. The primary anti-pp65 and R123 antibodies were diluted 1/500 and 1/200, respectively, in PBS containing 1 % foetal calf serum (PBSF). After incubation at room temperature for 1 h, the coverslips were washed at least six times with PBSF, then treated with both fluorescein isothiocyanate (FITC)-conjugated sheep anti-rabbit IgG (Sigma) and Cy5-conjugated goat anti-mouse IgG (Amersham), each diluted 1/200 in PBSF. After 30 min the coverslips were again washed with PBSF and mounted with AF1 (Citifluor). The coverslips were examined using a Zeiss LSM 510 confocal microscope system in conjunction with a Zeiss Axioplan 63x oil immersion objective lens (NA 1.4) and lasers with excitation lines at 488 and 633 nm. The two channels were scanned separately and the same settings maintained throughout. Captured images were exported and compiled using Adobe Photoshop.

Expression of functional UL15 and UL28 proteins

Virus yield complementation assays were performed in order to determine whether the parental plasmids used in these studies were capable of expressing functional UL15 and UL28 protein. Table 2 shows that in BHK cells transfected with pJM9 or pJM19 (which express untagged and pp65-tagged versions of full-length UL15, respectively) the yield of the UL15 null mutant S648 (titrated on clone 17 cells) was at least 500-fold greater than in control cells that received pUL28. Although a low level of recombinant virus capable of growth on Vero cells was detected (present in the S648 stock and/or generated by recombination with pJM9 or pJM19), the increased yields comprised predominantly mutant viruses, demonstrating that complementation had occurred in the presence of the two plasmids. Similarly, replication of the UL28 null mutant, gCB, occurred in cells transfected with pUL28 or pUL283, but not in control cells transfected with the vector pCMV10. These data indicate that the wild-type proteins expressed by both pJM9 and pUL28 are able to support virus replication and that the presence of the epitope tag on the protein encoded by pJM19 does not significantly alter the activity of the UL15 protein.

Table 2. Complementation of null mutants by HSV-1 UL15 and UL28 expression plasmids

BHK cells were transfected and superinfected with the indicated combination of plasmid and virus and the progeny were titrated on the cell lines shown (p.f.u./ml). ND, Not determined.

Expression of mutated UL28 polypeptides in mammalian cells

Plasmids containing deleted copies of the UL28 gene downstream of the HCMV major IE promoter were constructed as described in Methods and Table 1. The ability of the encoded proteins to function in DNA packaging was compared to wild-type UL28 encoded by pUL28 in a transient DNA packaging assay. BHK cells were co-transfected with the amplicon plasmid pSA1 and one of the plasmids encoding wild-type or mutated UL28, and superinfected with gCB. Total and packaged (DNase-resistant) DNA was prepared 20 h p.i. and the presence of replicated pSA1 was detected by Southern blotting and hybridization. Fig. 1 (b) shows that in each instance pSA1 replicated to a similar extent. Packaging of the replicated pSA1 DNA was undetectable in cells which received the control plasmid pCMV10 or mutant plasmids pUL281, pUL282, pUL284, pUL285 or pUL286. In contrast pSA1 was detectable in the DNase-resistant DNA fraction from cells transfected with pUL28 or pUL283. Both the wild-type protein and the pUL283 product lacking amino acids 465–477 support DNA encapsidation to a similar extent in this transient assay. In agreement with this pUL283 was also able to support the replication of gCB in the yield complementation assay (Table 2), while the other UL28 mutants did not (data not shown).

Fig. 1. Ability of mutated UL28 proteins to support DNA packaging. BHK cells were co-transfected with pSA1 and either a UL28-expressing plasmid or the vector pCMV10 as indicated and superinfected with gCB. At 20 h p.i. DNase-resistant DNA (a) and total DNA (b) were prepared and analysed as described in Methods. The positions of linear pSA1 molecules are indicated.

Co-immunoprecipitation of UL15 and UL28

Labelled extracts were prepared from Sf cells infected with AcUL28, AcUL15-pp65 or the two viruses in combination and precipitated with anti-pp65 antibody. Fig. 2 shows that UL15 was precipitated from both extracts prepared from cells which received AcUL15-pp65. In contrast, precipitation of UL28 was specifically dependent upon the presence of UL15. Since both UL15 and UL28 may be involved in binding to DNA, the experiment shown was performed in the presence of 50 µg/ml ethidium bromide throughout the precipitation and washing stages, which has been shown to selectively inhibit DNA-dependent protein associations (Lai & Herr, 1992). Subsequent experiments demonstrated that essentially identical results were obtained in the absence of ethidium bromide. Polyclonal antibody R123 against UL28 similarly specifically co-precipitated UL15 and UL28 from extracts of cells co-infected with AcUL28 and AcUL15, indicating that the presence of the pp65 epitope tag was not responsible for the observed interaction (data not shown). These results therefore support the earlier conclusion (Koslowski et al., 1999) that HSV-1 UL15 and UL28 interact.

Fig. 2. Co-immunoprecipitation of UL28 and UL15 proteins. Sf cells were infected with AcUL15-pp65 (15), AcUL28 (28) or the two viruses together and labelled with [³⁵S]methionine. Extracts were reacted with anti-pp65 antibody and the immunoprecipitates were analysed by SDS–PAGE alongside samples of total cell proteins and the starting extracts. Radioactivity in the dried gel was detected with a phosphorimager. The positions of the UL15 and UL28 proteins are indicated. The apparent faster migration of UL15 when precipitated in the absence of UL28 than in its presence is a feature of this particular gel and was not seen in other experiments.

Co-precipitation of mutant UL28 proteins with UL15

We next determined whether the mutated UL28 proteins could interact with UL15. The UL282, UL283, UL284, UL285 and UL286 proteins were expressed by recombinant baculoviruses and examined in co-immunoprecipitation experiments. Extracts prepared from Sf cells singly infected with these viruses or co-infected with AcUL15-pp65 were precipitated with anti-pp65 antibody and analysed by SDS–PAGE. Since the protein encoded by AcUL284 co-migrates with the tagged UL15 protein, the immunoprecipitated proteins were in some instances detected by Western blotting with antibody R123 against UL28. Fig. 3 (a) shows that wild-type UL28 and the UL282, UL283 and UL284 proteins were not detected by Western blotting in the control precipitates from singly infected cells but that all were specifically precipitated in the presence of UL15. Similarly, Fig. 3 (b) demonstrates that ³⁵S-labelled UL285 and UL286 were detected by phosphorimage analysis in the immunoprecipitates of extracts from cells mixedly infected with AcUL15-pp65 but not in the single infection controls. Specific co-precipitation of the UL282 and UL283 proteins with UL15 was also readily demonstrated by phosphorimage analysis of ³⁵S-labelled polypeptides (data not shown).

Fig. 3. Immunoprecipitation of mutant UL28 proteins with UL15. Sf cells were infected with the indicated recombinant baculoviruses [15, AcUL15-pp65; 28, AcUL28; 282, AcUL282; 283, AcUL283; 284, AcUL284; 285, AcUL285; 286, AcUL286] and labelled extracts were immunoprecipitated with anti-pp65 antibody. Total cellular proteins and the immunoprecipitates were resolved by SDS–PAGE. (a) The proteins were transferred to nylon membranes, UL28-related proteins were detected with antibody R123 and the membranes were photographed. (b) The gels were dried and radioactive proteins were detected with a phosphorimager.

These data therefore indicate that all five mutated proteins retain the ability to interact with UL15. The observation that both the UL285 and UL286 proteins interact suggests that at least two separate regions of UL28, located within amino acids 1–464 and 478–785 may independently contribute to binding. The less efficient co-precipitation of UL284 with UL15 (Fig. 3 a) was probably due to lower solubility, rather than a weaker interaction per se, although the actual contribution of amino acids 713–785 to binding remains to be determined.

UL15 molecules can interact with each other

A recombinant baculovirus expressing exon II of UL15 tagged with the pp65 epitope was used to test the possibility that UL15 molecules might interact with each other. Sf cells were infected either singly or in combination with AcUL15 and AcUL15E2-pp65, and extracts were prepared and immunoprecipitated with anti-pp65 antibody. Fig. 4 shows that the antibody precipitates the tagged exon II but not wild-type UL15 from singly infected cells. However, both the truncated and full-length proteins were detected following immunoprecipitation of the extract from mixedly infected cells. This demonstrates that UL15 molecules can interact not only with UL28 but also with each other, possibly enabling the formation of higher-order protein complexes.

Fig. 4. UL15 protein can interact with itself. Sf cells were infected with AcUL15 (15), AcUL15E2-pp65 (E2) or the two viruses together and labelled with [³⁵S]methionine. Extracts were reacted with anti-pp65 antibody and the immunoprecipitates were analysed by SDS–PAGE alongside samples of total cell proteins. Radioactivity in the dried gel was detected with a phosphorimager. The positions of the UL15 and exon II proteins are indicated.

Intracellular localization of UL15 and wild-type and mutant UL28 proteins

Initial experiments were performed in order to confirm that the co-expression of HSV-1 UL15 was necessary for the nuclear uptake of UL28 (Koslowski et al., 1999). Vero cells were transfected with pJM19 (encoding UL15-pp65) and pUL28 either separately or in combination using cationic liposomes. The cells were fixed and stained with a mixture of anti-pp65 and R123 primary antibodies followed by mixed Cy5- and FITC-conjugated secondary antibodies. Fig. 5 (a–c) shows that when expressed alone UL15-pp65 was specifically detected by excitation of the Cy5 fluor and localized to the nucleus. In contrast, UL28 alone was specifically detected by excitation of the FITC-conjugated antibody and exhibited a cytoplasmic localization (Fig. 5 d–f). In co-transfected cells both proteins localized to the nucleus (Fig. 5 g, h) where they exhibited a high degree of co-localization (merged image; Fig. 5 i). These data show that both proteins are readily detected in co-transfected cells and confirm that UL28 is only translocated to the nucleus in the presence of UL15.

Fig. 5. Intracellular localization of UL15 and UL28 proteins. Vero cells were transfected with pJM19 encoding UL15-pp65 (a–c), pUL28 (d–f) or both plasmids together (g–i). The cells were fixed, reacted with mixtures of the primary and secondary antibodies as described and examined by confocal microscopy. The three panels in each row (from left to right) show for the same field of cells Cy5 fluorescence (red, detecting UL15-pp65), FITC fluorescence (green, detecting UL28) and a merged image of the two.

Similar experiments were performed to investigate whether UL15 could translocate the mutated UL28 proteins to the nucleus (Fig. 6). In cells which received pUL281, pUL282, pUL283, pUL284, pUL285 or pUL286 alone the mutated UL28 protein in each instance exhibited a cytoplasmic localization similar to wild-type UL28 (Fig. 6 a, e, i, m, q, u). Co-expression with UL15 resulted in nuclear uptake of the pUL283, pUL285 and pUL286 proteins but not the other mutated UL28 polypeptides (Fig. 6 b, f, j, n, r, v). As expected, the nuclear UL28 proteins showed a high degree of co-localization with UL15 (Fig. 6 k, l, s, t, w, x). Surprisingly, in the presence of the other three UL28 mutants (UL281, UL282 and UL284) UL15 was retained in the cytoplasm, but again extensively co-localized with the UL28 protein (Fig. 6 c, d, g, h, o, p).

Fig. 6. Intracellular localization of UL15 and mutated UL28 proteins. Vero cells were transfected with pUL281, pUL282, pUL283, pUL284, pUL285 or pUL286 either alone (a, e, i, m, q, u) or together with pJM19 encoding UL15-pp65 (b–d, f–h, j–l, n–p, r–t, v–x), and the expressed proteins were detected by confocal microscopy as described in the legend to Fig. 5. The left panel of each row shows representative staining of UL28 proteins with FITC in the singly transfected cells. The next three panels show FITC staining (UL28 proteins), Cy5 staining (UL15-pp65) and a merged image of the two for the same field of co-transfected cells. (y, z) Merged images of FITC staining (UL28 proteins) and Cy5 staining (UL9) in Vero cells co-transfected with pUL28 and pE9 (y) or pUL281 and pE9 (z).

In order to exclude the possibility that these mutated UL28 proteins might be having a non-specific inhibitory effect on the nuclear uptake of proteins, they were co-expressed with HSV-1 UL9 which, like UL15, is known to be efficiently translocated into the nucleus when expressed alone (Malik et al., 1996). UL9 protein was detected with monoclonal antibody 13924 and Cy5-conjugated secondary antibody, and UL28 was detected with R123 and FITC-conjugated secondary antibody. Fig. 6 (y) and (z) indicate that in cells co-transfected with pE9, expressing UL9 protein (Stow et al., 1993), and either pUL28 or pUL281, respectively, localization of UL9 to the nucleus was unaffected, whilst the UL28 protein remained in the cytoplasm. Identical results were obtained in co-transfections with pUL282 and pUL284 (data not shown), indicating that UL15 is specifically retained in the cytoplasm of cells co-expressing these three mutated UL28 proteins. Each of the six mutated UL28 proteins therefore retains the ability to interact with UL15, as indicated either by an ability to retain UL15 in the cytoplasm or to be translocated with it into the nucleus.

Koslowski et al. (1999) previously demonstrated that HSV-1 UL28 required the presence of UL15 in order to be translocated into the nucleus and that the two proteins co-purified from HSV-1-infected cells, apparently as a heterodimer. These data provided strong evidence for an interaction between the two proteins. In this paper we have presented two additional independent lines of evidence to support this hypothesis. First, UL28 specifically co-precipitated with UL15 from extracts of Sf cells infected with recombinant baculoviruses (Fig. 2). Second, we have extended the previous immunofluorescence data by demonstrating a high degree of co-localization of the two proteins in cells in which they are both expressed (Fig. 5). The significance of these observations is enhanced by the demonstration that the cloned UL15 and UL28 genes used in these experiments can support DNA packaging and virus growth (Fig. 1 and Table 2).

Taken together with the earlier demonstration of an interaction between PRV UL28 and HSV-1 UL15 (Koslowski et al., 1997), and the genetic support for an interaction between the homologous proteins of HCMV (Underwood et al., 1998; Krosky et al., 1998) there now seems little doubt that the two proteins establish a functional interaction and that this is likely to be conserved throughout the herpesvirus family. Circumstantial evidence, outlined in Introduction, suggests that the two proteins may function in a similar way to the bacteriophage terminases during packaging, but this remains to be verified experimentally. Our demonstration that UL15 molecules have the ability to interact with one another (Fig. 4) is not inconsistent with this suggestion. In fact the heterodimeric bacteriophage terminases probably form multimers as complexes containing the DNA to be packaged, terminase enzyme and preformed capsid are assembled (Fujisawa & Morita, 1997; Catalano, 2000). It will therefore be of interest to determine whether UL15 and UL28 can generate larger complexes during packaging or if the UL15–UL15 and UL15–UL28 interactions are mutually exclusive.

This paper reports the first examination of the ability of HSV-1 UL28 mutants to interact with HSV-1 UL15. The intracellular localization of PRV UL28 mutants was previously examined by immunofluorescence in cells co-infected with a PRV UL28 null mutant or co-transfected with an HSV-1 UL28-expressing plasmid (Koslowski et al., 1997). These two approaches yielded consistent results, although it should be noted that evidence for the formation of a functional complex between the PRV and HSV-1 proteins is lacking. The results of our immunoprecipitation and immunofluorescence experiments (Figs 3 and 6) provide complementary lines of evidence that five of the UL28 mutants (UL282, UL283, UL284, UL285 and UL286) remain able to interact with HSV-1 UL15. A similar conclusion was reached for the sixth mutant, UL281, based only on the fluorescence study. Since the regions of UL28 contained within the UL285 and UL286 proteins are non-overlapping (amino acids 1–464 and 478–785, respectively) it would appear that at least two separate regions of HSV-1 UL28 must be able to interact independently with UL15. This result differs from the study with PRV UL28 mutants in which the C-terminal 155 amino acids were required for binding to UL15 of either PRV or HSV-1 (Koslowski et al., 1997). A possible explanation is that the PRV protein contains interacting sequences corresponding to those in the C-terminal but not the N-terminal portion of the HSV-1 protein. However, the ability of several of our UL28 mutants to retain UL15 in the cytoplasm raises the alternative possibility that the PRV protein containing amino acids 1–569 formed a complex with UL15 which remained in the cytoplasm and was not detected because the cells were not co-stained with an antibody that would recognize the latter protein. Interestingly, the presence of separate regions of UL28 able to interact with UL15 might possibly contribute to the two proteins forming multimeric assemblies, as proposed above.

Inspection of alignments of the amino acid sequences of homologues of the UL28 protein encoded by alpha-, beta- and gammaherpesviruses reveals that the regions of highest conservation are confined to amino acids 1–427 and 496–785 of the HSV-1 sequence. The intervening region not only shows poor sequence conservation but also differs significantly in length between different viruses, suggesting that it might possibly serve as a 'spacer' between separate domains of the protein. Of the six mutants tested only UL283, which contains the smallest deletion (13 amino acids), was able to support virus growth and DNA packaging (Table 1 and Fig. 1) and interestingly its lesion is entirely within this poorly conserved region.

Although the ability of the UL281, UL282 and UL284 proteins to retain UL15 in the cytoplasm constitutes strong evidence for an interaction, the mechanism by which this occurs is not clear. Since these proteins did not inhibit UL9 nuclear localization it is unlikely that they cause a non-specific inhibition of nuclear transport. It is possible that the failure of their complexes with UL15 to enter the nucleus results from misfolded regions masking a UL15 nuclear localization signal or causing decreased solubility of the complex in the cytoplasm. The presence of distinct domains within UL28 probably contributes to the mechanism by which these three proteins retain UL15 in the cytoplasm, since in each instance one of the two postulated domains remains intact and potentially able to fold correctly to provide an interface for the protein–protein interaction, even though the remaining portions may be misfolded.

The inability of these three proteins to complement the null mutant gCB for growth or DNA packaging can also be explained by their failure to enter the nucleus, but it remains likely that the regions deleted also contain residues directly involved in the packaging process. The phenotypes of the UL285 and UL286 proteins indicate that sequences from within both the putative domains contribute to the nuclear function of UL28. In this regard it is interesting to note that residues 197–225 of the HSV-1 protein contain a motif (CX₂CX₈NXGX₁₁CXH) which is conserved throughout the mammalian and avian herpesviruses and may represent a metal ion-binding region. It is hoped that site-directed mutagenesis of this and other regions of HSV-1 UL28 will shed further light upon the functions and interactions of this protein during DNA packaging.

We thank Fred Homa, Joel Baines and Andrew Davison for providing virus null mutants, complementing cell lines and the UL15 'cDNA' fragment. James McVicar, the recipient of a ROPA award from the Medical Research Council, provided excellent technical assistance and we are grateful to Duncan McGeoch for helpful comments on the manuscript.

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