MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
Author for correspondence: Nigel Stow. Fax +44 141 337 2236. e-mail n.stow{at}vir.gla.ac.uk
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Abstract |
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Introduction |
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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, 1998a
; 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, 1998b
).
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.
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Methods |
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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.
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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 138785 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 acidglycine 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 Eagles 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 oriS 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 acidglycine 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 ASepharose beads, washed and the proteins separated by SDSPAGE. 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 Dulbeccos 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.
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Results |
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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.
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Discussion |
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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 UL15UL15 and UL15UL28 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 (UL28
2, UL28
3, UL28
4, UL28
5 and UL28
6) remain able to interact with HSV-1 UL15. A similar conclusion was reached for the sixth mutant, UL28
1, based only on the fluorescence study. Since the regions of UL28 contained within the UL28
5 and UL28
6 proteins are non-overlapping (amino acids 1464 and 478785, 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 1569 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 1427 and 496785 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, UL28
2 and UL28
4 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 proteinprotein 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 UL28
6 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 197225 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.
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Acknowledgments |
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References |
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Addison, C., Rixon, F. J. & Preston, V. G.(1990). Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids.Journal of General Virology71, 2377-2384.[Abstract]
Ali, M. A., Forghani, B. & Cantin, E. M.(1996). Characterization of an essential HSV-1 protein encoded by the UL25 gene reported to be involved in virus penetration and capsid assembly.Virology216, 278-283.[Medline]
Baines, J. D., Cunningham, C., Nalwanga, D. & Davison, A.(1997). The UL15 gene of herpes simplex virus type 1 contains within its second exon a novel open reading frame that is translated in frame with the UL15 gene product. Journal of Virology71, 2666-2673.[Abstract]
Bishop, D. H. L.(1992). Baculovirus expression vectors.Seminars in Virology3, 253-264.
Bogner, E., Radsak, K. & Stinski, M. F.(1998). The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity.Journal of Virology72, 2259-2264.
Catalano, C. E.(2000). The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.Cellular and Molecular Life Sciences 57, 128-148.[Medline]
Catalano, C. E., Cue, D. & Feiss, M.(1995). Virus DNA packaging: the strategy used by phage lambda.Molecular Microbiology16, 1075-1086.[Medline]
Cavalcoli, J. D., Baghian, A., Homa, F. L. & Kousoulas, K. G.(1993). Resolution of genotypic and phenotypic properties of herpes simplex virus type 1 temperature-sensitive mutant (KOS) tsZ47: evidence for allelic complementation in the UL28 gene.Virology197, 23-34.[Medline]
Davison, A. J.(1992). Channel catfish virus: a new type of herpesvirus.Virology186, 9-14.[Medline]
Dolan, A., Arbuckle, M. & McGeoch, D. J.(1991). Sequence analysis of the splice junction in the transcript of herpes simplex virus type 1 UL15.Virus Research20, 97-104.[Medline]
Fujisawa, H. & Morita, M.(1997). Phage DNA packaging. Genes to Cells2, 537-545.
Goshima, F., Watanabe, D., Takakuwa, H., Wada, K., Daikoku, T., Yamada, M. & Nishiyama, Y.(2000). Herpes simplex virus UL17 protein is associated with B capsids and colocalizes with ICP35 and VP5 in infected cells.Archives of Virology145, 417-426.[Medline]
Homa, F. L. & Brown, J. C.(1997). Capsid assembly and DNA packaging in herpes simplex virus.Reviews in Medical Virology7, 107-122.[Medline]
Kitts, P. A., Ayres, M. D. & Possee, R. D.(1990). Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.Nucleic Acids Research18, 5667-5672.[Abstract]
Koslowski, K. M., Shaver, P. R., Wang, X.-Y., Tenney, D. J. & Pederson, N. E.(1997). The pseudorabies virus UL28 protein enters the nucleus after coexpression with the herpes simplex virus UL15 protein.Journal of Virology71, 9118-9123.[Abstract]
Koslowski, K. M., Shaver, P. R., Casey, J. T.II, Wilson, T., Yamanaka, G. Y., Sheaffer, A. K., Tenney, D. J. & Pederson, N. E.(1999). Physical and functional interactions between the herpes simplex virus UL15 and UL28 DNA cleavage and packaging proteins.Journal of Virology73, 1704-1707.
Krosky, P. M., Underwood, M. R., Turk, S. R., Feng, K. W.-H., Jain, R. K., Ptak, R. G., Westerman, A. C., Biron, K. K., Townsend, L. B. & Drach, J. C.(1998). Resistance of human cytomegalovirus to benzimidazole ribonucleosides maps to two open reading frames: UL89 and UL56.Journal of Virology72, 4721-4728.
Lai, J.-S. & Herr, W.(1992). Ethidium bromide provides a simple test for identifying genuine DNA-independent protein associations.Proceedings of the National Academy of Sciences, USA89, 6958-6962.[Abstract]
Lamberti, C. & Weller, S. K.(1998). The herpes simplex virus type 1 cleavage/packaging protein, UL32, is involved in efficient localization of capsids to replication compartments.Journal of Virology72, 2463-2473.
Livingstone, C. & Jones, I.(1989). Baculovirus expression vectors with single strand capability.Nucleic Acids Research17, 2366.[Medline]
McGeoch, D. J., Dalrymple, M. A., Davison, A. J., Dolan, A., Frame, M. C., McNab, D., Perry, L. J., Scott, J. E. & Taylor, P.(1988). The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1.Journal of General Virology69, 1531-1574.[Abstract]
McLean, G. W., Abbotts, A. P., Parry, M. E., Marsden, H. S. & Stow, N. D.(1994). The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein.Journal of General Virology75, 2699-2706.[Abstract]
McNab, A. R., Desai, P., Person, S., Roof, L. L., Thomsen, D. R., Newcomb, W. W., Brown, J. C. & Homa, F. L.(1998). The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA.Journal of Virology72, 1060-1070.
Malik, A. K., Shao, L., Shanley, J. D. & Weller, S. K.(1996). Intracellular localization of the herpes simplex virus type 1 origin binding protein, UL9.Virology224, 380-389.[Medline]
Nasseri, M. & Mocarski, E. S.(1988). The cleavage recognition signal is contained within sequences surrounding an aa junction in herpes simplex virus DNA.Virology167, 25-30.[Medline]
Patel, A. H. & Maclean, J. B.(1995). The product of the UL6 gene of herpes simplex virus type 1 is associated with virus capsids.Virology206, 465-478.[Medline]
Poon, A. P. W. & Roizman, B.(1993). Characterization of a temperature-sensitive mutant of the UL15 open reading frame of herpes simplex virus 1.Journal of Virology67, 4497-4503.[Abstract]
Reynolds, A. E., Fan, Y. & Baines, J. D.(2000). Characterization of the UL33 gene product of herpes simplex virus 1.Virology266, 310-318.[Medline]
Rixon, F. J.(1993). Structure and assembly of herpesviruses.Seminars in Virology4, 135-144.
Rosenthal, K. S., Leuther, M. D. & Baines, B. G.(1984). Herpes simplex virus binding and entry modulate cell surface protein mobility. Journal of Virology49, 980-983.[Medline]
Salmon, B. & Baines, J. D.(1998). Herpes simplex virus DNA cleavage and packaging: association of multiple forms of UL15-encoded proteins with B capsids requires at least the UL6, UL17 and UL28 genes.Journal of Virology72, 3045-3050.
Salmon, B., Cunningham, C., Davison, A. J., Harris, W. J. & Baines, J. D.(1998). The herpes simplex virus type 1 UL17 gene encodes virion tegument proteins that are required for cleavage and packaging of viral DNA.Journal of Virology72, 3779-3788.
Stow, N. D.(1998). Transient assays for HSV origin and replication protein function. In Herpes Simplex Virus Protocols, pp. 215-226. Edited by S. M. Brown & A. R. MacLean. Totowa, NJ:Humana Press.
Stow, N. D. & McMonagle, E. C.(1983). Characterization of the TRS/IRS origin of DNA replication of herpes simplex virus type 1.Virology130, 427-438.[Medline]
Stow, N. D. & Wilkie, N. M.(1976). An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA.Journal of General Virology33, 447-458.[Abstract]
Stow, N. D., McMonagle, E. C. & Davison, A. J.(1983). Fragments from both termini of the herpes simplex virus type 1 genome contain signals required for the encapsidation of viral DNA.Nucleic Acids Research11, 8205-8220.[Abstract]
Stow, N. D., Hammarsten, O., Arbuckle, M. I. & Elias, P.(1993). Inhibition of herpes simplex virus type 1 DNA replication by mutant forms of the origin-binding protein. Virology196, 413-418.[Medline]
Stow, N. D., Brown, G., Cross, A. M. & Abbotts, A. P.(1998). Identification of residues within the herpes simplex virus type 1 origin-binding protein that contribute to sequence-specific DNA binding.Virology240, 183-192.[Medline]
Taus, N. S. & Baines, J. D.(1998). Herpes simplex virus DNA cleavage/packaging: the UL28 gene encodes a minor component of B capsids.Virology252, 443-449.[Medline]
Tengelsen, L. A., Pederson, N. E., Shaver, P. R., Wathen, M. W. & Homa, F. L.(1993). Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants.Journal of Virology67, 3470-3480.[Abstract]
Towbin, H., Staehelin, T. & Gordon, J.(1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, USA76, 4350-4354.[Abstract]
Underwood, M. R., Harvey, R. J., Stanat, S. C., Hemphill, M. L., Miller, T., Drach, J. C., Townsend, L. B. & Biron, K. K.(1998). Inhibition of human cytomegalovirus DNA maturation by a benzimidazole ribonucleoside is mediated through the UL89 gene product.Journal of Virology72, 717-725.
Yu, D. & Weller, S. K.(1998a). Herpes simplex virus type 1 cleavage and packaging proteins UL15 and UL28 are associated with B but not C capsids during packaging.Journal of Virology72, 7428-7439.
Yu, D. & Weller, S. K.(1998b). Genetic analysis of the UL15 gene locus for the putative terminase of herpes simplex virus type 1.Virology243, 32-44.[Medline]
Yu, D., Sheaffer, A. K., Tenney, D. J. & Weller, S. K.(1997). Characterization of ICP6::lacZ insertion mutants of the UL15 gene of herpes simplex virus type 1 reveals the translation of two proteins. Journal of Virology71, 2656-2665.[Abstract]
Received 27 July 2000;
accepted 6 September 2000.